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Antemortem Diagnosis of Coxiellosis in a Blue and Gold Macaw (Ara ararauna).

Abstract: A 15-year-old female blue and gold macaw (Ara ararauna) was presented for evaluation after being found laterally recumbent, reluctant to move, and lethargic. Results of a complete blood count showed an increased number of immature heterophils with increased cytoplasmic basophilia and degranulation and the presence of a left shift. Radiographs and a computed tomography scan were performed and revealed a markedly enlarged spleen. An ultrasound-guided fine-needle aspirate of the spleen was submitted for cytologic examination and aerobic bacterial culture. While the culture revealed no growth, cytologic examination identified mononuclear phagocytes with cytoplasmic vacuoles containing structures consistent with bacteria. Pan-bacterial 16S rRNA polymerase chain reaction of the splenic sample followed by direct sequencing identified a Coxiella-like agent identical to one previously isolated in the liver of a golden-mantled rosella (Platycercus eximius). Phylogenetic analysis shows that avian coxiellosis agents and Coxiella burnetii, the agent of Q fever, represent 2 independent events of development of vertebrate pathogenicity in this group of tick endosymbionts. This report suggests diagnostic and treatment directions for coxiellosis in avian patients and indicates where further study is needed.

Key words: splenomegaly, coxiellosis, Coxiella-like endosymbiont, antemortem, avian, blue and gold macaw, Ara ararauna

Clinical Report

A 15-year-old female blue and gold macaw (Ara ararauna) was presented, after being found laterally recumbent, reluctant to move, and lethargic. It was acquired by a private owner approximately 1.5 years before presentation and was quarantined for 90 days before introduction to other birds at the multispecies breeding facility in which it was housed. This bird was housed in a large (2.1-m X 1.2-m X 4.3-m) metal outdoor enclosure with 1 male blue and gold macaw. Its diet consisted of seeds, pellets, nuts, fruits, and vegetables. It had no change in appetite before presentation. Initial evaluation determined the bird to be in moderate body condition (915 g) with chronic osteoarthritis of the left elbow joint, a caudodorsal coelomic mass, and generalized ataxia.

Blood tests were performed, and while results of the serum biochemical analysis were unremarkable, results of the complete blood count showed an increased number of immature heterophils (0.85 X [10.sup.3] cells/pL), cells that occur rarely in avian blood. (1) Toxicity, evidenced by increased cytoplasmic basophilia and degranulation, was also noted among heterophils and, with the presence of a left shift in this series, indicated an inflammatory leukogram. Radiographs and a computed tomography scan were performed, and the palpable coelomic mass was found to be consistent with a grossly enlarged spleen (Fig 1). Additionally, a mildly distended proventriculus, increased medullary opacity of the right humerus, and osteoarthritis of the left elbow joint were seen. A sample of the spleen collected via ultrasound-guided fine-needle aspirate was submitted for cytologic examination and aerobic bacterial culture. On cytologic examination, nucleated cells consisted predominantly of a heterogeneous lymphoid population and hematopoietic precursors (Fig 2). Lymphoid cells consisted of small and intermediate to large lymphocytes admixed with low to moderate numbers of plasma cells, consistent with a reactive population. Low numbers of mononuclear phagocytes were also present and were occasionally noted to have a cytoplasmic vacuole containing small, thin, variably staining, linear to pleomorphic structures, consistent with bacteria (Fig 3). Similar structures could also be found focally on the slides in extracellular aggregates (Fig 3). Given the morphology of the organisms and association with the mononuclear phagocytes, mycobacteriosis was considered, and a Ziehl-Neelsen acid-fast stain was performed on one of the previously stained cytologic preparations. The structures did not stain with this technique, making infection with a Mycobacterium species less likely. The bird was discharged from the hospital 8 days later with no definitive diagnosis and was started on a regimen of meloxicam (0.3 mg/kg PO q24h), enrofioxacin (15 mg/kg PO q24h), and azithromycin (10 mg/kg PO q48h).

There was no growth of aerobic bacterial or mycobacterial organisms on culture from the splenic aspirate. DNA was extracted from a sample of the splenic aspirate by using a commercial kit (DNEasy tissue kit, Qiagen, Valencia, CA, USA). Nested polymerase chain reaction (PCR) amplification of the bacterial 16S rRNA gene using consensus primers was done by previously described methods. (2) The PCR product was resolved in a 1% agarose gel, and the band was excised and purified using a QIAquick gel extraction kit (Qiagen). Direct sequencing was performed by using the Big-Dye Terminator Kit (Perkin-Elmer, Branchburg, NJ, USA) and analyzed on ABI automated DNA sequencers at the University of Florida Interdisciplinary Center for Biotechnology Research Sequencing Facilities. All products were sequenced in both directions. Primer sequences were edited out before further analyses. The product was 863 base pairs after primers were edited out. Results of the PCR assay followed by direct sequencing determined the unidentified bacteria in the splenic sample to be a Coxiella-like species identical to one previously isolated from the liver of a golden-mantled rosella (Platycercus eximius). (3) The sequence was submitted to GenBank under accession number KX611832.

The sequences were compared to those in GenBank (National Center for Biotechnology Information, Bethesda, MD, USA), EMBL (Cambridge, UK), and Data Bank of Japan (Mishima, Shizuoka, Japan) databases using BLASTN. (4) Predicted homologous 860-870 nucleotide sequences of representative gammaproteobacterial 16S rRNA were downloaded from GenBank and aligned using MAFFT, as previously described. (5) Bayesian analysis was performed using MrBayes (6) on the CIPRES server 7 with gamma-distributed rate variation and a proportion of invariant sites, and a general time-reversible model. Thioalbus denitrificans (GenBank accession no. NR_122087) was used as an outgroup. Four chains were run and statistical convergence was assessed by looking at the standard deviation of split frequencies and potential scale reduction factors of parameters. The first 10% of 2 000 000 iterations were discarded as a burn-in, based on examination of trends of the log probability versus generation. Two independent analyses were performed to avoid entrapment on local optima. The Bayesian tree is shown in Figure 4.

Maximum likelihood analysis of the alignment was performed by using RAxML on the CIPRES server as previously described, (8) using a gamma-distributed rate variation, a proportion of invariant sites, and a general time-reversible model. Again, Thioalbus denitrificans (GenBank accession no. NR_122087) was used as an outgroup. To test the strength of the tree topology, bootstrap analysis was used (1000 resamplings).[degrees] The maximum likelihood bootstrap values are shown on the Bayesian tree (Fig 4).

After receiving PCR results (on day 25), an approximately 200-day course of doxycycline (20 mg/kg PO ql2h) was prescribed in accordance with the primary treatment for chronic Coxiella infection in people (ie, Query [Q] fever). (10) Although treatment for coxiellosis has yet to be reported in birds, the efficacy of doxycycline in the treatment of Q fever is well documented and its use in avian patients is extensive. (11) However, the bird died the next day, before initiating doxycycline therapy. It was subsequently submitted for necropsy.

Postmortem examination revealed severe splenomegaly with multifocal areas of necrosis, hemorrhage, and capsular tears. Associated with the splenic capsular tears, there was a moderate amount of hemorrhage and clots within the coelomic cavity. Histologic examination revealed a lymphohistiocytic and heterophilic splenitis with multifocal thrombosis and necrosis, multifocal necrotizing hepatitis, and multifocal exocrine pancreas necrosis. In addition, within splenic and hepatic macrophages and acinar cells of the exocrine pancreas, a single, clear vacuole peripheralized the nuclei. The vacuoles contained small amounts of eosinophilic granular material that stained with periodic acid-Schiff histochemical stain (Fig 5). Similar postmortem gross and histologic findings have been previously described with Coxiella-like infection in birds. (3,12) Splenic hemorrhage was determined to be the proximate cause of death.

Discussion

Avian coxiellosis appears to be an emerging infectious disease with significant mortality among captive species of bird. The characterization of the clinical signs and pathology associated with this disease are of utmost importance in the diagnosis and treatment of this disease, benefitting avian veterinarians and researchers alike. Fatal coxiellosis has been reported in several instances in 9 species of captive birds. These case reports included postmortem diagnoses of infection by Coxiella-like organisms in 11 total birds, encompassing 8 psittacine species and 1 toucan (Ramphastos toco). (3,12,13) These included a hawk-headed parrot (Deroptyus accipitrinus), a golden-mantled rosella, 2 cockatiels (Nymphicus hollandicus), a canary-winged parakeet (Brotogeris versicolurus chiriri), a kakariki (Cyanoramphus novaezelandiae), a blue and gold macaw, (3) 3 Swainson's Blue Mountain rainbow lorikeets (Trichoglossus haematodus moluccanus), (13) and an eclectus parrot (Eclectus roratus). (12) These birds presented with nonspecific clinical signs including lethargy, weakness, and neurologic abnormalities. Gross necropsy revealed varying levels of emaciation, splenomegaly, and hepatomegaly. Histologic findings for many of the birds included Coxiella-like bacteria within macrophages of the spleen, liver, bone marrow, kidneys, and adrenals, identified using 16S bacterial PCR and electron microscopy. (3) Granulomatous encephalitis and myocarditis were found in the lorikeets (13) and eclectus parrot, (12) respectively. The only history available concerning the blue and gold macaw was that it was male and died suddenly without premonitory signs. Postmortem examination revealed that it weighed 808 g and that the spleen was severely enlarged and ruptured, with hemorrhage in the coelom. (3) Further pathologic findings specific to this bird were unavailable.

The antemortem identification of a Coxiella in this bird has many implications regarding diagnosis and treatment of this disease. There is a wide range of clinical signs that may be associated with avian coxiellosis, including lethargy, decreased body condition, cardiac abnormalities, or neurologic issues. Results of blood tests may be indicative of chronic infection. Imaging may reveal varying levels of splenomegaly, hepatomegaly, or cardiac abnormalities associated with endocarditis or myocarditis. Other diagnostic procedures to be considered include ultrasound-guided fine-needle aspirate of the organ(s) of concern and subsequent cytologic examination and bacterial culture. Because clinical signs may be nonspecific, specific testing is essential for the early diagnosis of coxiellosis. In this clinical report, splenic hemorrhage was determined to be the proximate cause of death. While splenic rupture has been previously reported in avian coxiellosis cases, (3) and more than a week passed between splenic aspiration and rupture, the potential role of iatrogenic injury caused by the fine-needle aspirate must also be considered.

The best available model with significant data for diagnosis and treatment of avian coxiellosis is Q fever. Coxiella is a genus of bacteria in the phylum Proteobacteria, class Gammaproteobacteria, order Legionellales, family Coxiellales. They are gram-negative organisms and, like most Legionellales, are obligate intracellular bacteria. (14) The best-known member of the genus is Coxiella burnetii, the causative agent of Q fever, a worldwide zoonotic disease that infects the phagolysosome of macrophages. This disease is known to spread via direct contact or aerosol inhalation of infected bodily fluids of ruminants, especially placental fluids; however, transmission by infected ticks or other animals (eg, rats) has been speculated. (15,16) In people, the acute form may manifest itself as headache, fever, and malaise while the chronic form is more severe and may result in endocarditis. (17) Diagnosis can be obtained using a combination of serology and PCR. (14,18) Treatment requires a long-term course of doxycycline. (17)

While cattle, sheep, and goats may be the most recognized hosts of C burnetii, a number of other mammals and birds exist as possible reservoirs. (18) Although antibodies to this bacterium have been observed in wild bird populations, (19,20) infection by C burnetii is not known to be pathogenic in birds. Until recently, this bacterium was the only named species within the genus Coxiella. However, the identification of Coxiella cheraxi in crayfish (Cherax quadricarinatus) (21) and other Coxiella-like endosymbionts suggests that there is far more genetic diversity within the genus than previously perceived. These Coxiella-like endosymbionts have been widely described in over 40 species of ticks, including those that are ectoparasites of seabirds (18,22,23) and monk parakeet (Myiopsitta mon achus) colonies. (24) Although exposure of birds to C burnetii is not known to result in clinical disease, some Coxiella-like organisms appear to have a different pathogenicity.

Current methods of Q fever diagnosis in people include serologic testing and PCR. (14,18) Immunofluorescence assays are the most common serologic test used to assess antibody response to C burnetii in people. (18) The results of these tests are variable depending on the length of time since infection, the extent of humoral versus cellular response, and whether the subject is acutely or chronically infected. These tests can use either serum or whole blood, making sample choice and accessibility simple. However, serologic tests are sometimes limited in their value because test results may not indicate current infection, as antibodies may still be present from past or resolved infections. Currently there are no isolates or characterized antigens of avian Coxiella species, so serologic assays for avian coxiellosis are not currently available. With the increased awareness of avian coxiellosis, serologic testing using immunofluorescence assays potentially could be developed and utilized in a similar fashion as in human medicine to efficiently and noninvasively diagnose disease specific to these organisms.

Q fever may also be diagnosed via PCR assays. (14) Depending on the assay and sample population, sensitivities and specificities vary. In one 7-year study of Q fever diagnosis in people, a quantitative real-time PCR was shown to have approximately 71% sensitivity and up to 100% specificity with the highest sensitivity on cardiac valvular samples and lower values for blood serum and urine samples. (25) However, other studies have found C burnetii DNA to be undetectable in serum at times during the acute antibody response. (26) As Coxiella are highly cell-associated bacteria, serum is not expected to be a good sample, and one study found PCR positives in buffy coats when serum and whole blood were negative. The diagnosis of coxiellosis in animals may not be as straightforward, as tissue sample availability and test sensitivity may be decreased. Samples such as uterine fluid, milk, and vaginal mucus have shown unreliable levels of sensitivity and specificity in dairy goats. (27) Because of this, when concerning avian patients in a clinical setting, further studies on comparative diagnostic testing of different sample types are needed to make appropriate recommendations. In this case, a splenic aspirate was used for pan-bacterial 16S rRNA PCR diagnosis. However, PCR testing using universal eubacterial primers and sequencing is more time consuming than a PCR assay specific for this avian coxiellosis agent would be, such as quantitative PCR assay. In this case, 2 weeks passed between initiation of PCR assay and the diagnosis of coxiellosis. The bird died before doxycycline therapy could be started. A quantitative PCR assay specific for this avian Coxiella species may have yielded results sooner, accelerated the diagnosis, and provided a more timely opportunity for treatment.

Treatment of Q fever in people should be initiated based on clinical signs and relevant risk factors but should not be delayed while waiting for laboratory results. (17) The standard treatment includes administration of doxycycline for a course of 2 weeks for acute illness and 18-24 months for chronic illness. (10) For avian patients with suspected coxiellosis, possible risk factors may include outdoor housing, exposure to ticks, exposure to other infected animals, or immunosuppression. In this case, the macaw was housed outdoors (with suspected exposure to ticks), in the same cage as a male blue and gold macaw, and around other birds. However, these risk factors alone did not immediately indicate coxiellosis. The most prominent physical abnormality in this case was the marked splenomegaly; this may be an indication for clinicians to consider coxiellosis in birds. This bird was initially treated with nonsteroidal anti-inflammatory drugs and antibiotics before PCR results were available. When the PCR results indicated coxiellosis, doxycycline was prescribed; however, the bird died before treatment. In future avian coxiellosis cases, doxycycline is a reasonable choice, but studies are needed to evaluate its efficacy.

It is presently unknown if avian coxiellosis is directly transmitted or arthropod vectored. Q fever is often directly transmitted; however, as can be seen in the phylogenetic tree (Fig 4), the most closely related organisms are endosymbionts of ticks that have been documented as having essential and mutualistic relationships. (28,29) Some even perform metabolic functions such as vitamin biosynthesis. (30) Each endosymbiont has a tick species with which it is closely linked; these organisms are not known to be pathogenic and are instead thought to have evolved as maternally inherited and beneficial endosymbionts. (24) Endosymbionts of special interest include those of avian ectoparasites or nest parasites. One endosymbiont found in Ornithodoros capensis, a tick commonly found in seabird nests, is closely related to C burnetii. (23) Another closely related endosymbiont has been identified in an Argas monachus, a tick found in a monk parakeet colony. (24) These endosymbionts possibly have the capacity to cause disease. Although disease due to these agents in birds has not been identified, the lack of specificity of clinical signs associated with avian coxiellosis, the lack of available diagnostic testing, and the limited surveillance that has been done in wild bird colonies has made disease recognition quite difficult. Further study of coxiellosis in wild bird populations is indicated. Additionally, the epidemiologic impact of coxiellosis in migratory bird species should also be considered; avian migrations often involve multiple continents, and infections can rapidly become cosmopolitan. Coxiella burnetii is classified as a category B bioterrorism agent (31); given this, the zoonotic potential of avian coxiellosis merits investigation.

The agents that have been recognized in avian coxiellosis as described above, the agent from the blue and gold macaw described in this case, and a related agent found in a hawk-headed parrot are more divergent from each other than is seen between C burnetii strains. These agents are not very close to C burnetii on the Coxiella tree and likely represent a separate adaptation of a tick endosymbiont into a vertebrate pathogen. The greater divergence between characterized avian coxiellosis agents may imply a longer history of this clade as vertebrate pathogens than C burnetii.

Coxiella burnetii has a larger overall genome size and subsequently a higher number of genes when compared with related tick endosymbionts, suggesting that C burnetii has additional evolutionary strategies for pathogenicity and therefore spread and survival. (24) Comparative genomic analysis of C burnetii, the avian coxiellosis agent, and related tick endosymbionts may identify virulence genes and would be helpful in predicting which related tick endosymbionts present the greatest risk to become pathogenic in vertebrates.

References

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Alison J. Flanders, Justin F. Rosenberg, DVM, Marjorie Bercier, DMV, Mary K. Leissinger, DVM, Dipl ACVP, Laura J. Black, DVM, Robson F. Giglio, DVM, PhD, Serena L. M. Craft, DVM, Dipl ACVP, Whitney M. Zoll, DVM, DACVP, April L. Childress, and James F. X. Wellehan, DVM, PhD, Dipl ACZM, Dipl ACVM, Dipl ECZM

From the Departments of Comparative, Diagnostic, and Population Medicine (Flanders, Rosenberg, Bercier, Lessinger, Black. Craft. Zoll. Shildress, Wellehan) and Small Animal Clinical Sciences (Giglio), College of Veterinary Medicine. University of Florida, 2015 SW 16th Avenue. Gainesville. FL 32610, USA.

Caption: Figure 1. Right lateral radiograph of a blue and gold macaw that was presented after being found laterally recumbent, reluctant to move, and lethargic. The markedly enlarged spleen is indicated with an arrow.

Caption: Figure 2. Fine-needle aspirate of an intracoelomic mass in the macaw described in Figure 1. A heterogeneous lymphoid population is present (thick arrows) in moderate number admixed with few myeloid precursors (thin arrow) and moderate amounts of blood. Wright-Giemsa stain. Bar = 50 [micro]m.

Caption: Figure 3. Fine-needle aspirate of an intracoelomic mass in the macaw described in Figure 1. Thin pleomorphic to elongate bacteria are present extracellularly (top left panel) and in cytoplasmic vacuoles within mononuclear phagocytes (top right and bottom panels). Wright-Giemsa stain. Bar = 20 [micro]m.

Caption: Figure 4. Bayesian phylogenetic tree of MAFFT alignment of homologous 860-870 base pairs of representative gammaproteobacterial 16S rRNA sequences. Thioalbus denitrificans (GenBank accession no. NR_122087) was used as the outgroup. Confidence of the tree topology obtained is shown by Bayesian posterior probabilities to the left of the slash or above, and maximum likelihood bootstrap values are to the right or below. The avian pathogenic clade is marked in blue, C burnetii is in red, and the tick endosymbiont from monk parrot nests is in green.

Caption: Figure 5. Photomicrograph of histologic tissue samples of the macaw described in Figure 1. (A) Spleen. Macrophages are frequently distended by large clear vacuoles that variably contain flocculent eosinophilic material (arrows). Hematoxylin and eosin stain. Bar = 50 [micro]m. (B) Liver. A hepatic macrophage distended by a clear vacuole that contains eosinophilic granular material and 2 lightly basophilic slightly plump to elongate bacteria (arrow). Hematoxylin and eosin stain. Bar = 50 [micro]m.
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Title Annotation:Clinical Report
Author:Flanders, Alison J.; Rosenberg, Justin F.; Bercier, Marjorie; Leissinger, Mary K.; Black, Laura J.;
Publication:Journal of Avian Medicine and Surgery
Date:Dec 1, 2017
Words:4270
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