On July 17, 2014, a 27-day-old male cassowary (Casuarius casuarius) chick (case 1) was found weak and recumbent in its enclosure at a zoo in southeastern Virginia. The bird was manually restrained for physical examination and venipuncture. It subsequently developed abnormally increased respiratory effort and was weak and mentally obtunded. Whole-body radiographs were obtained and revealed a loss of serosal detail in the coelomic cavity and lungs with an abnormally decreased volume bilaterally and abnormally increased opacity, which was most prominent near the hilum (Figure 1). The chick was treated with midazolam (0.29 mg/kg [0.13 mg/lb], IM, once) as a prophylactic measure against the neurological signs that were anticipated. The chick also received ceftiofur hydrochloride (20 mg/kg [9 mg/lb], IM, once), furosemide (1.8 mg/kg [0.82 mg/lb], IM, once), enrofloxacin (5 mg/kg [2.27 mg/lb], IM, once), and isotonic saline (0.9% NaCl) solution (50 mL, SC) on the basis of treatment recommendations for critically ill exotic birds.1 A blood sample was obtained for a serum biochemical analysis and CBC. Clinicopathologic abnormalities included hyperuricemia (27.7 mg/dL; reference range,2 2.0 to 16.5 mg/dL), hypocalcemia (3.8 mg/dL; reference range,2 8.3 to 14.2 mg/dL), hyperphosphatemia (13.9 mg/dL; reference range,2 2.9 to 11.7 mg/dL), and abnormally high AST (3,379 U/L; reference range,2 243 to 811 U/L), GGT (121 U/L; reference value,2 27 U/L), and creatine kinase (2,538 U/L; reference range,2 192 to 1,246 U/L) activities. It is likely that amylase (6,479 U/L) and LDH (> 3,600 U/L) activities were also abnormally increased, but reference ranges for those enzymes in cassowaries have not been established. Microscopic examination of a blood smear revealed numerous heterophils with signs of marked acute toxicosis including cytoplasmic basophilia, moderate degranulation, and abnormal granules. All other CBC variables were within their respective reference ranges. Abnormalities identified during plasma electrophoresis included a low total protein concentration and low albumin-to-globulin ratio characterized by a mild to moderate increase in β globulins, which was indicative of an acute infection. The chick was kept in a warm incubator with oxygen supplementation and was found dead approximately 12 hours after the initial physical examination.
Right lateral whole-body radiographic image of a 27-day-old male southern cassowary (Casuarius casuarius) chick (case 1) with clinical disease from EEEV. The radiograph reveals loss of coelomic detail and a decrease in lung volume accompanied by an increase in the soft-tissue opacity of the lungs (arrow).
Citation: Journal of the American Veterinary Medical Association 249, 3; 10.2460/javma.249.3.319
A necropsy was performed. Abnormal gross findings included enlarged and friable kidneys with a white exudate present on cut surfaces and a white mucofibrinous exudate adhered to the walls of the ventriculus; coelomic effusion was not evident. Histologic abnormalities included necrosuppurative hepatitis (Figure 2), nephritis, ventriculitis, mild multifocal necrosuppurative interstitial pneumonia, and multisystemic pinpoint necrosis that was suggestive of a viral infection. Aerobic bacterial culture of the contents of the proventriculus, ventriculus, and intestines yielded heavy growth of normal fecal flora; Salmonella spp were not isolated.
Photomicrograph of a section of liver from the cassowary chick of Figure 1. Notice the perivascular necrosis (arrows) with small numbers of lymphocytes, macrophages, and heterophils. H&E stain; bar = 20 µm. See Figure 1 for remainder of key.
Citation: Journal of the American Veterinary Medical Association 249, 3; 10.2460/javma.249.3.319
Fresh frozen brain tissue was sent for evaluation of EEEV to the Diagnostic Center for Population and Animal Health at Michigan State University in Lansing, Michigan. The RNA was extracted from the brain tissue by use of a monophasic solution of phenol and guanidine isothiocyanatea in accordance with the manufacturer's directions for evaluation in 2 separate RT-PCR assays. The first assay targeted a 112-base pair region of the viral genome that encodes the EEEV capsid protein.3 The second assay (an in-house assay) targeted a 207-base pair region of the viral genome that encodes portions of the viral E3 protein and viral E2 envelope glycoprotein. Presence of EEEV was confirmed by nucleic acid sequencing of the E3 protein-E2 envelope glycoprotein amplicon at the Research Technology Support Facility at Michigan State University. The derived sequences were analyzed with a bioinformatic search tool.b Results indicated that the RNA isolated from the brain of case 1 had high similarity (99% to 100%) with sequences of the North American strain of EEEV published in GenBank.
On the same day that case 1 was identified as ill, 2 other 27-day-old male cassowary chicks (cases 2 and 3) were found dead in the same enclosure. Both chicks had been clinically normal the previous day. Necropsies were performed on both chicks, and the findings were identical. When the carcasses were found, they were in good condition albeit in a state of pronounced rigor mortis. The head and neck of each carcass were in prominent dorsiflexion. Grossly, there was copious yellow mucoid fluid in the coelomic cavity, brown mucoid fluid in the intestines, and swollen kidneys. Thick, white mucoid material was adhered to the mucosa of the ventriculus and proventriculus. The lungs were dark red bilaterally. Body fat stores appeared to be adequate, which was indicative of acute death. Histologic abnormalities included acute periportal hepatic necrosis with lymphohistiocytic perivascular inflammation, necrotizing vasculitis of the spleen and ventriculus, multifocal pulmonary hemorrhage, perivascular edema and tubular casts in the kidneys, and mild lymphohistiocytic perivascular inflammation of the cerebellum. Collectively, the gross and histologic findings suggested that a viral infection was likely. Aerobic bacterial culture of the intestinal fluid yielded heavy growth of normal intestinal flora; Salmonella spp were not identified. Results of RT-PCR assays conducted on fresh frozen brain tissue were identical to those for case 1.
In August 2014, approximately 2 weeks after the death of cases 1, 2, and 3, the 23-year-old dam (case 4) of those chicks was observed to be lethargic. The bird had been vaccinated with a killed polyvalent equine encephalomyelitis–West Nile virus vaccinec (1.0 mL, IM via a dart) approximately 1 week earlier. It was treated with enrofloxacin1 (10 mg/kg [4.5 mg/lb], IM, q 24 h via a dart). The next day the bird was more lethargic, appeared inappetent, and had mild ataxia. Treatment with enrofloxacin was continued, and meloxicam (0.18 mg/kg [0.08 mg/lb], IM, once) was also administered. The following day, the bird was found sternally recumbent and extremely weak. It was easily restrained manually for physical examination and venipuncture. Abnormal physical examination findings included hypopyon bilaterally, erythematous oral mucosa, an increase in respiratory effort, and moderate dehydration as evidenced by the presence of stringy, mucoid saliva in the oral cavity. Notable clinicopathologic findings included hyperuricemia (74.6 mg/dL), hypochloremia (89 mEq/L; reference range,2 90 to 106 mEq/L), hyperphosphatemia (12.2 mg/dL), abnormally increased AST (2,969 U/L) and GGT (163 U/L) activities, and a suspected abnormally increased LDH activity (> 3,600 U/L). Results of a CBC revealed an absolute monocytosis (3,946 monocytes/µL; reference range,2 0 to 3,619 monocytes/µL); many monocytes had basophilic granules and vacuolization. There was also a moderate number of heterophils with signs of marked acute toxicity such as cytoplasmic basophilia, moderate degranulation, abnormal granules, and cytoplasmic vacuolization. The plasma β globulin concentration was increased from reference limits, which was indicative of acute inflammation.
The bird was administered isotonic saline solution (1 L, SC), enrofloxacin (10 mg/kg, IM), and ampicillin1 (12 mg/kg [5.45 mg/lb], IM, once). It continued to deteriorate throughout the day and began to have tonic-clonic seizures that became progressively worse. The bird died shortly after a grand mal seizure that lasted several minutes.
A necropsy was performed immediately after death, and the carcass was subjectively assessed as abnormally warm. Gross abnormalities included the presence of extensive hemorrhage along the medial aspect of the left leg (likely the result of trauma during seizure activity); a large volume of thick, viscous, yellow fluid throughout the coelomic cavity; swollen kidneys with serosal hemorrhages; multifocal serosal hemorrhages throughout the intestines; and congested mesenteric blood vessels. The spleen and liver both appeared swollen and friable. The left ovary was covered with active follicles including 1 large thick black follicle that was approximately 5 cm in diameter. Histologic abnormalities included moderate multifocal mineralization of the kidney and a necrotic yolk sac tumor. Sections of kidney, liver, and spleen contained similar but rare inflammatory infiltrates with tissue necrosis comparable to that observed in the previous cases. The heart and some sections of neural tissue did not contain notable microscopic lesions. However, neuronal satellitosis was present within the cerebral gray matter, and neurons within the brainstem contained intracytoplasmic pigment consistent with lipofuscin. Cytologic examination of fluid (suspected hypopyon) aspirated from an eye revealed marked heterophilic inflammation with likely bacterial infection. Aerobic bacterial culture of the interior aspect of the necrotic ovarian follicle yielded large numbers of Staphylococcus xylosus. Results of RT-PCR assays on fresh frozen brain tissue were positive for EEEV.
The death of 4 cassowaries from EEEV infection within a 2-week period in southeastern Virginia prompted us to seek out other instances of EEEV infection in southern cassowaries. We uncovered 2 additional cases from a conservation center in Florida.
In December 1996, a 15-year-old male cassowary (case 5) at a conservation center in Florida was observed with weakness and ataxia. The bird had been obtained from California approximately 1 month prior and was vaccinated with a killed polyvalent equine encephalitis vaccinec at that time. Physical examination revealed that the bird appeared stunted with poor feather condition and a grade 4/6 systolic heart murmur. Clinicopathologic abnormalities included leukocytosis (35,900 WBCs/µL; reference range,4 10,724 to 24,836 WBCs/µL), hypocalcemia (9.6 mg/dL; reference range,4 10.1 to 12.5 mg/dL), hyperproteinemia (7.5 g/dL; reference range,4 4.6 to 6.2 g/dL), hyperglobulinemia (5.7 g/dL; reference range,4 1 to 3 g/dL), hypoalbuminemia (1.8 g/dL; reference range,4 2.4 to 4 g/dL), and abnormally increased AST activity (805 U/L; reference range,4 246 to 776 U/L). Results of plasma electrophoresis revealed a suspected hyper–β globulinemia (3.17 g/dL; reference range unavailable for this species) and hyperproteinemia (7.2 g/dL; reference range unavailable for this species), which were indicative of acute inflammation. Serum was sent to 2 separate laboratories for determination of anti-EEEV antibody titers by use of a hemagglutination inhibition assay. The serum anti-EEEV antibody titer determined by the Florida State Diagnostic Laboratory in Kissimmee, Florida was 1:640, and that determined by the US Geological Survey National Wildlife Health Center in Madison, Wisconsin, was 1:2,560. The bird was treated with supportive care and enrofloxacin1 (8.7 mg/kg [3.95 mg/lb], PO, q 12 h) for 14 days. Its clinical condition remained unchanged following completion of the enrofloxacin regimen.
In March 1997, the bird was examined because of lameness in the right leg. At that time, it received a booster vaccination of a killed polyvalent equine encephalitis vaccinec and a blood sample was obtained for a CBC, serum biochemical analysis, and determination of anti-EEEV antibody titers. Results of the CBC revealed a persistent leukocytosis (37,900 WBCs/µL). Clinicopathologic abnormalities included mild hyperchloremia (108 mEq/L), hypoalbuminemia (1.0 g/dL), hypoproteinemia (2.4 g/dL), and hyperkalemia (4.0 mEq/L; reference range,4 2.1 to 3.7 g/dL). Results of plasma electrophoresis revealed a suspected hyper–β globulinemia (1.36 g/dL). The serum anti-EEEV antibody titer was 1:640 as determined by the Florida State Diagnostic Laboratory and 1:1,280 as determined by the US Geological Survey National Wildlife Health Center.
The bird was found dead unexpectedly in July 1997. Gross abnormalities observed during necropsy included yellow fluid in the coelomic cavity, an enlarged liver, and the presence of green mucoid fluid in the intestines. The air sacs appeared opacified and the lungs were congested bilaterally. The pericardium was thickened and fibrotic. The left atrium was enlarged, and there was fibrinous dysplasia of the atrioventricular valves of the heart. Histologic abnormalities included renal tubular degeneration and necrosis, mild to moderate subacute enteritis, moderate subacute hepatitis, and severe diffuse subacute bilateral pulmonary congestion and hemorrhage in addition to systemic vasculitis, proventricular myositis, and orchitis, which were indicative of viral or bacterial septicemia. Virus isolation performed on specimens of the brain, spleen, and liver yielded negative results for EEEV. Results of immunohistochemical staining of sections of the brain and spleen for EEEV antigen were likewise negative. At that time, serum anti-EEEV antibody titers > 1:640 as determined by hemagglutination inhibition were considered diagnostic for EEEV infection; a RT-PCR assay for detection of EEEV RNA to confirm infection was not available.
In April 2009, a 9-month-old female cassowary (case 6) at a conservation center in Florida developed lethargy, weakness, ataxia, and obtunded mentation. A CBC revealed leukocytosis (32,600 WBCs/µL). Clinicopathologic abnormalities included hyperglycemia (262 mg/dL; reference range,4 141 to 223 mg/dL), hypouricemia (2.9 mg/dL; reference range,5 4.2 to 28.8 mg/dL), hypophosphatemia (4.1 mg/dL: reference range,4 4.6 to 7 mg/dL), and a suspected abnormally increased GGT activity (50 U/L; laboratory reference4 used had no reference range available for that species). The bird was treated with enrofloxacin1 (10 mg/kg, SC, once), meloxicam1 (0.10 mg/kg [0.045 mg/lb], SC, once), and lactated Ringer solution (400 mL, SC). The next day the bird was extremely weak, mildly dehydrated, and minimally responsive. Results of a follow-up CBC revealed a leukocytosis (42,500 WBCs/µL) with an absolute heterophilia (34,430 heterophils/µL; reference range,4 2,321 to 6,767 heterophils/µL) and degenerative left shift. Clinicopathologic abnormalities included hypouricemia (3.9 mg/dL), hyperglobulinemia (4.2 g/dL), and hyperproteinemia (7.0 g/dL). The treatment regimen was changed to amikacin1 (5.0 mg/kg, IV, q 12 h), ampicillin1 (50 mg/kg [22.7 mg/lb], IV, q 12 h), and meloxicam1 (0.12 mg/kg [0.054 mg/lb], PO, q 24 h). The bird's clinical condition worsened to include paresis of both legs, dyspnea and abnormal respiratory signs, pyrexia, and regurgitation. It died on the afternoon of the third day after it was first observed ill.
A necropsy was performed soon after death and revealed that the carcass was in excellent condition with adequate stores of body fat. Gross abnormalities included splenomegaly and congestion of the liver and pancreas. The left lung was dark red to purple in color and consolidated, whereas the right lung was red and moist and floated in formalin. Regurgitated food material was found around the glottis, and the tracheal mucosa appeared inflamed. Histologic abnormalities included subacute aspiration pneumonia bilaterally, lymphohistiocytic pneumonia, severe heterophilic air sacculitis, pericarditis, hepatitis, lymphocytic enteritis, and mild lymphocytic encephalitis. Serum neutralization testing revealed that the bird had a > 10-fold increase in anti-EEEV antibody titer (> 1:100) between the day that the clinical signs were first observed and death, which was indicative of an acute EEEV infection. Specimens of brain tissue were submitted to the National Veterinary Services Laboratory in Ames, Iowa, for RT-PCR assay, and results were positive for EEEV. The bird had been vaccinated with a killed polyvalent equine encephalitis vaccinec at 2, 4, 6, 8, and 10 weeks old.
Discussion
Eastern equine encephalitis virus is an important cause of encephalitis in horses and humans.6–9 Eastern equine encephalitis virus is an arbovirus that belongs to the genus Alphavirus in the family Togaviridae and is endemic in the eastern United States.10,11 Infection with EEEV is typically not fatal for native birds in endemic areas but is highly fatal for humans, horses, and exotic birds.10–12 The virus is maintained in a cycle between black-tailed mosquitos (Culiseta melanura) and avian hosts.5,10,12,13 Numerous species of free-living passerine and columbiform birds serve as natural hosts for the virus.7,14
In birds native to North America, EEEV has been associated with the death of a great egret and birds in the orders Gruidae and Icteridae.12 Disease caused by EEEV has also been reported in various introduced and domestic avian species such as ring-necked pheasants, chickens, psittacines, glossy ibises, Pekin ducks, pigeons, rock doves, emus, ostriches, Chukar partridges, and house sparrows.12,13,15 The mortality rate varies among species; whooping cranes, in particular, are prone to die from EEEV infections.12 In birds, infection with EEEV often results in encephalitis and meningitis, although viscerotropism characterized by vasculitis, hepatic and splenic necrosis, and hemorrhage is not uncommon,12 and pathological lesions vary among species. In mammals, EEEV infection typically results in neurotropism.7
The southern cassowary is a large ratite native to New Guinea and northeastern Australia.5,16 The species is classified as vulnerable on the basis of current conservation status.17 There are currently 40 southern cassowaries held in Association of Zoos and Aquariums–accredited zoos in North America.d In the present report, we described the clinical, hematologic, and pathological findings for 6 southern cassowaries that died subsequent to infection with EEEV. Four of those birds (cases 1 through 4) died at a Virginia zoo within a 2-week period. This prompted the zoo to vaccinate the 3 remaining cassowaries in its collection, and those birds remained clinically unaffected. Interestingly, the sire of cases 1 through 3, which lived in the same enclosure as those chicks, remained clinically unaffected. The acute nature of the deaths of those 3 chicks likely prevented them from shedding large amounts of the virus into the environment.7 No other wild or captive animals at the zoo or humans in the local area became ill or died from EEEV infection during that time. Immediately after the death of cases 1 through 4 in 2014, the Ratite Taxon Advisory Group issued a formal recommendation to vaccinate all cassowaries with a killed equine polyvalent vaccinec at 2, 4, 6, 8, and 10 weeks with booster vaccines administered to adults annually before the start of mosquito season.e
Several outbreaks of EEEV infection resulting in high morbidity and mortality rates in captive emus (Dromaius novahollandiae), another ratite species, have been reported.7,9,13,18 In emus, infection with EEEV is associated with a rapid onset of clinical signs and high mortality rates. Infected emus are commonly found dead without premonitory signs but may also have signs of depression, disseminated intravascular coagulation, hemorrhagic diarrhea, and emesis of blood-stained ingesta.7,13,19 Pathological lesions in EEEV-infected emus often include necrotizing hepatitis and splenitis; the CNS is generally unaffected.7 In other ratite species, pathological lesions most commonly associated with EEEV infection include viscerotropism and necrohemorrhagic enteritis of the mucosal layer.6 In the cassowaries of the present report, hemorrhagic diarrhea and emesis were not commonly observed, and similar to emus, neurotropism was largely lacking.
Results of 1 study19 indicate that EEEV-infected emus develop high titers of the virus in their blood, making them important virus reservoirs. They also shed the virus in secretions and excretions, which makes them infectious for sympatric birds and humans and provides an opportunity for transmission of the virus in the absence of mosquitos.19 Therefore, isolation of suspected EEEV-infected animals is recommended.
Outbreaks of EEEV infection have occurred in captive whooping cranes20 and a flock of African penguins.21 Clinical signs of EEEV infection in African penguins include anorexia, antisocial behavior, lethargy, vomiting, and regurgitation. In some infected penguins, those signs progress to severe ataxia, recumbency, and seizures.21 Common clinicopathologic abnormalities in EEEV-infected penguins include persistent heterophilic leukocytosis and monocytosis, anemia, and hyperglobulinemia.21 During 1 outbreak,21 14 of 22 penguins seroconverted to EEEV, and 13 of those 14 penguins survived with intensive supportive care. For EEEV-infected penguins, the mean time required for clinical signs to resolve was 12 days, and the mean time required for clinicopathologic abnormalities to resolve was > 30 days.21 Approximately a third of the penguins that survived EEEV infection had permanent residual neurologic deficits most commonly characterized by mild ataxia.21
Histologic lesions observed in birds infected with EEEV are not pathognomonic for the virus. Various other diagnostic testing modalities such as hemagglutination inhibition, immunohistochemistry, and virus isolation have historically been used to confirm that EEEV is the etiologic agent responsible for the lesions.8 However, RT-PCR testing is now widely available and is highly sensitive and specific for the detection of EEEV RNA in fresh tissues.12
Results of 1 study19 indicate that emus vaccinated with a commercially available killed polyvalent equine encephalitis vaccine develop anti-EEEV antibodies titers that are protective against disease. A monovalent vaccine against EEEV also appears to be efficacious for protection against naturally acquired infection in whooping cranes when administered IM.10,19 However, case 6 of the present report was vaccinated with a polyvalent equine encephalitis vaccine 5 times as a chick and still died subsequent to EEEV infection, which indicated that vaccination against EEEV is not always protective against disease.
Eastern equine encephalitis virus is an important zoonotic pathogen. Approximately one third of human patients that become infected with EEEV and develop clinical signs of the disease die, and many of those who survive have permanent neurologic damage.5,14 Various mosquito species such as Aedes, Coquillettidia, and Culex that feed on both birds and humans can transmit EEEV to humans. Infection caused by EEEV is a reportable disease in most states and should also be reported to the CDC. In the United States, approximately 5 to 10 human patients develop clinical disease as a result of EEEV infection annually; most cases originate in the Atlantic and Gulf Coast states from late spring to early fall.5
The cassowaries infected with EEEV described in the present report developed similar clinical signs of disease, which included weakness, lethargy, and anorexia. Three of the 6 EEEV-infected birds died unexpectedly without any premonitory signs. Commonly reported clinicopathologic findings included leukocytosis, hyperuricemia, and abnormally increased activities of liver enzymes (AST, GGT, and LDH). Coelomic effusion was a common necropsy finding. Although the histologic findings varied among the 6 cases, viscerotropism was frequently evident. Vasculitis of the coelomic organs, particularly the liver and spleen, was a hallmark lesion in all cases, which was consistent with histologic findings in other EEEV-infected avian species including emus. However, unlike EEEV-infected emus, hemorrhagic lesions within the gastrointestinal tract were not observed in the EEEV-infected cassowaries described in this report. Eastern equine encephalitis virus causes high morbidity and mortality rates in southern cassowaries, and clinical disease might be reduced or prevented by vaccination, isolation of ill birds, and mosquito control strategies.
Acknowledgments
The authors thank Sara Hallagher for technical support.
ABBREVIATIONS
| AST | Aspartate aminotransferase EEEV Eastern equine encephalitis virus |
| GGT | γ glutamyltransferase |
| LDH | Lactate dehydrogenase |
| RT-PCR | Reverse-transcriptase PCR |
Footnotes
Trizol reagent, Life Technologies Corp, Carlsbad, Calif.
BLAST, National Center for Biotechnology Information, National Institutes of Health, Bethesda, Md. Available at: blast.ncbi.nlm.nih.gov/. Accessed Dec 22, 2014.
Vetera EWT+WNV, Boehringer Ingelheim Vetmedica Inc, St Joseph, Mo.
LaGreco N, Zoological Society of San Diego, San Diego, Calif: Personal communication, 2015.
Valitutto M, Staten Island Zoo, Staten Island, NY: Personal communication, 2015.
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