Wildlife

Veterinary Pathology
50(2) 291-298
Mycoplasma Corogypsi–Associated ª The Author(s) 2012
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Polyarthritis and Tenosynovitis in Black DOI: 10.1177/0300985812457791
vet.sagepub.com
Vultures (Coragyps atratus)

A. J. Van Wettere1,2, D. H. Ley2, D. E. Scott3, H. D. Buckanoff4, and

L. A. Degernes5

Abstract
Three wild American black vultures (Coragyps atratus) were presented to rehabilitation centers with swelling of multiple joints,
including elbows, stifles, hocks, and carpal joints, and of the gastrocnemius tendons. Cytological examination of the joint fluid exudate
indicated heterophilic arthritis. Radiographic examination in 2 vultures demonstrated periarticular soft tissue swelling in both birds
and irregular articular surfaces with subchondral bone erosion in both elbows in 1 bird. Prolonged antibiotic therapy administered in
2 birds did not improve the clinical signs. Necropsy and histological examination demonstrated a chronic lymphoplasmacytic arthritis
involving multiple joints and gastrocnemius tenosynovitis. Articular lesions varied in severity and ranged from moderate synovitis and
cartilage erosion and fibrillation to severe synovitis, diffuse cartilage ulceration, subchondral bone loss and/or sclerosis, pannus,
synovial cysts, and epiphyseal osteomyelitis. No walled bacteria were observed or isolated from the joints. However, mycoplasmas
polymerase chain reactions were positive in at least 1 affected joint from each bird. Mycoplasmas were isolated from joints of 1
vulture that did not receive antibiotic therapy. Sequencing of 16S rRNA gene amplicons from joint samples and the mycoplasma
isolate identified Mycoplasma corogypsi in 2 vultures and was suggestive in the third vulture. Mycoplasma corogypsi identification was
confirmed by sequencing the 16S-23S intergenic spacer region of mycoplasma isolates. This report provides further evidence that M.
corogypsi is a likely cause of arthritis and tenosynovitis in American black vultures. Cases of arthritis and tenosynovitis in New World
vultures should be investigated for presence of Mycoplasma spp, especially M. corogypsi.

Keywords
avian, arthritis, black vulture, Coragyps atratus, Mycoplasma corogypsi, polymerase chain reaction, bacterial isolation, tenosynovitis

Members of the genus Mycoplasma are known causes of arthritis
and synovitis in mammals, birds, and reptiles.28 In avian species,
Mycoplasma spp–induced arthritis and tenosynovitis are well
documented in galliformes where infection with Mycoplasma
synoviae or, less commonly, Mycoplasma gallisepticum causes
articular and tendon lesions in chickens and turkeys, affecting
infected populations with increased morbidity and mortality.28
Mycoplasma spp have been isolated from several species of
wild and captive birds of prey.11,14,16,18–20,22,23 Associations
between respiratory signs or disease and Mycoplasma spp in
raptors have been hypothesized in multiple reports.2,9,11,18,20–
23
However, most reports did not include a comprehensive
clinical and pathological workup to support these assump-
tions.2,5,11,15,23,26 There are only 2 instances of associations
between Mycoplasma spp infection and musculoskeletal
disease in raptors. Mycoplasma buteonis was associated with
a perosis-type skeletal deformity in a saker falcon (Falco
cherrug) nestling, and M. corogypsi was associated with poly-
arthritis in 1 American black vulture (Coragyps atratus).5,26
In New World vultures, the only Mycoplasma species
reported is Mycoplasma corogypsi. This organism was
isolated from a pododermatitis lesion, and its 16S ribosomal
RNA (rRNA) gene was detected in a case of polyarthritis,
both times in an American black vulture.20,26 Here, we
report the clinical presentation, gross pathology, histology,
isolation, and molecular diagnosis of 3 cases of polyarthritis
and tenosynovitis in American black vultures associated
with M. corogypsi.
1
Center for Comparative Medicine and Translational Research, College of
Veterinary Medicine, North Carolina State University, Raleigh, NC
2
Department of Population Health and Pathobiology, College of Veterinary
Medicine, North Carolina State University, Raleigh, NC
3
Carolina Raptor Center, Huntersville, NC
4
The Valerie H. Schindler Wildlife Rehabilitation Center, Asheboro, NC
5
Department of Clinical Sciences, College of Veterinary Medicine, North
Carolina State University, Raleigh, NC
Corresponding Author:
Laurel A. Degernes, Department of Clinical Sciences, College of Veterinary
Medicine, 1060 William Moore Drive, North Carolina State University, Raleigh,
NC 27607.
Email: laurel_degernes@ncsu.edu
292
Material and Methods
Case Histories and Clinical Findings
Vulture No. 1. A wild adult female American black vulture
was presented to a wildlife rehabilitation center for inability
to fly. The bird was alert and thin (1631 g) and had bilateral soft
tissue swelling over both elbow joints. Radiographs revealed
irregular articular surfaces and subchondral bone erosion of the
humeral/ulnar joints. Treatments included analgesics (meloxi-
cam 0.5 mg/kg orally every 12 hours, Metacam, Boehringer
Ingelheim Vetmedica Inc., St. Joseph, Missouri) and antibiotics
(trimethoprim and sulfamethoxazole 50 mg/kg orally every 12
hours, Hi-Tech Pharmacal, Amityville, New York) for 19 days.
The elbow swelling did not improve, and a complete blood cell
count (CBC) done at 19 days showed normal hematocrit3 and
revealed leukocytosis (estimated white blood cell count [WBC]
was 108,728 cells/ml, with 61% heterophils, 32% lymphocytes,
2% eosinophils, and 5% monocytes). No WBC reference range
was available for the American black vulture in the literature,
so a reference range for the turkey vulture (Cathartes aura) was
used: WBC 20,100 cells/ml (range 10,500–31,900 cells/ml).3,27
Antibiotic treatment was switched to enrofloxacin (15 mg/kg
every 12 hours orally, Baytril, Bayer Healthcare LLC, Shawnee
Mission, Kansas) and amoxicillin/clavulanic acid (125 mg/kg
every 12 hours orally, Clavamox, Pfizer Animal Health, Exton,
Pennsylvania). Between days 26 and 39, firm subcutaneous
abscesses proximal to both elbows were lanced and flushed 4
times while the bird was under isoflurane anesthesia. The CBC
results on day 26 revealed anemia (Packed cell volume [PCV]
33%) and leukocytosis (43,120 cells/ml with 78% heterophils,
18% lymphocytes, and 4% monocytes). Clindamycin (100
mg/kg every 12 hours orally, Ranbaxy Pharmaceuticals, Jack-
sonville, Florida) was added to the treatment regimen. At day
39, bilateral joint crepitation was palpated in both elbows. The
bird was deemed nonreleasable, and all treatments were dis-
continued except meloxicam until the bird was presented to the
North Carolina State University College of Veterinary Medi-
cine (NCSU-CVM) for diagnostic evaluation 50 days after
admission.
Vulture No. 2. A nonflighted wild juvenile male American
black vulture was lethargic and thin (1717 g) and had bilateral
soft tissue swelling over elbow joints, hock joints, distal
tibiotarsi, proximal tarsometatarsi, and digits. No bony
changes were noted radiographically. The CBC revealed ane-
mia (PCV 27%) and leukocytosis (WBC 58,800 cells/ml with
87% heterophils and 13% lymphocytes). Treatments included
doxycycline (75 mg orally every 12 hours, Doxycycline
Hyclate, West-Ward Pharmaceuticals, Eatontown, New Jer-
sey) and meloxicam. At day 40, the vulture was clinically
lame on the left leg, and copious amounts of thick caseous
exudate were surgically flushed from the left elbow joint dur-
ing isoflurane anesthesia. The estimated WBC remained ele-
vated at 46,577 cells/ml (53% heterophils, 38% lymphocytes,
5% eosinophils, and 4% monocytes). Based upon the lack of
response to treatment, the bird was deemed nonreleasable.
Veterinary Pathology 50(2)
Antibiotic therapy was discontinued, but analgesic therapy
(meloxicam and tramadol [20 mg/kg every 12 hours orally,
Amneal Pharmaceuticals, Hauppauge, New York]) was admi-
nistered until the bird was submitted to NCSU-CVM 51 days
after admission.
Vulture No. 3. A nonflighted wild juvenile male American
black vulture was weak, dehydrated, and thin (1347 g) and had
bilateral soft tissue swelling over elbow, carpal, and hock joints
and distal tibiotarsi. The bird was mildly anemic (PCV 38%)
and had mild leukocytosis (WBC 23,400 cells/ml with 56% het-
erophils, 34% lymphocytes, 2% eosinophils, and 8% mono-
cytes). Based upon multiple joint involvement and the poor
results obtained with antibiotic therapy in vulture Nos. 1 and
2, treatment was not attempted and euthanasia was recom-
mended. Supportive care (fluids and feeding) and analgesic
therapy (meloxicam) were administered until the bird was sub-
mitted to NCSU-CVM 2 days after admission.
Gross Pathological, Cytological, and Histopathological
Examination
The 3 black vultures were euthanatized with an intravenous
overdose of sodium barbiturate while under isoflurane anesthe-
sia and submitted immediately for postmortem examination to
NCSU-CVM. Representative tissue samples of the trachea,
lung, liver, spleen, proventriculus, ventriculus, intestinal tract,
pancreas, heart, kidney, brain, peripheral nerves, skeletal
muscle, adrenal glands, thyroid glands, bone marrow, thymus,
testis, ovary, and elbow and hock joints were fixed in 10% neu-
tral buffered formalin, processed, and embedded in paraffin
according to routine histological techniques. The bone samples
were decalcified with formic acid solution (20%) before
paraffin embedding. Sections 5-mm thick were stained with
hematoxylin and eosin and examined by light microscopy. Sec-
tions of elbow and hock joints were also stained with Giemsa,
Gram’s, and Gomori methenamine sulfate (GMS) stains
according to standard methods. Direct smears from joint fluid
were made and stained with Wright’s-Giemsa stain for
cytological examination.
Toxicology
Samples of liver (vulture Nos. 1 and 3) were collected during
necropsy, saved frozen, and submitted to the Pennsylvania
Animal Diagnostic Laboratory System–New Bolton Center
(Kennett Square, Pennsylvania) for lead concentration analysis
using atomic absorption spectroscopy. Blood lead level for vul-
ture No. 2 was analyzed antemortem (LeadCare analyzer, ESA
Biosciences Inc., Chelmsford, Massachusetts).
Bacteriology
Swabs from heart blood and both elbow joints (vulture Nos. 1
and 3), liver (vulture No. 1), and heart blood, left elbow joint,
and left hock joint (vulture No. 2) were submitted to Rollins
Wettere et al
Animal Disease Diagnostic Laboratory (North Carolina
Department of Agriculture & Consumer Services, Raleigh,
North Carolina) for bacterial isolation under aerobic conditions
(vulture No. 1) and aerobic and anaerobic conditions (vulture
Nos. 2 and 3). A joint fluid aspirate (vulture Nos. 1 and 3) and
a swab from the articular and synovial surfaces of the right and
left elbow (vulture Nos. 1, 2 and 3) and right elbow and carpal
joints (vulture No. 3) and from hock joints, trachea, and left
caudal thoracic air sac (vulture Nos. 2 and 3) were submitted
for mycoplasma culture and polymerase chain reaction (PCR)
to the NCSU-CVM Mycoplasma Diagnostic and Research
Laboratory. At necropsy, samples from vulture No. 1 were
inoculated to Remel M5 transport medium (Thermo Fisher Sci-
entific, Remel Products, Lenexa, Kansas). Upon arrival at the
laboratory, within 2 hours of sample collections, 200 ml from
each was transferred to 2 ml of Frey’s broth medium with
15% (v/v) swine serum (FMS) and 1% (v/v) Fungizone (250
mg/ml amphotericin B, GIBCO Cat. No. 15290-018) for
mycoplasma culture.10 Samples from vulture Nos. 2 and 3 were
inoculated directly to 2 ml of FMS with Fungizone for myco-
plasma culture. Inoculated broth cultures were incubated in
humidified air at 37 C with periodic transfer of aliquots to
additional FMS broth and agar media with incubation for up
to 3 weeks, during which broth and agar media were examined
daily for evidence of mycoplasmal growth.
DNA Extraction, PCR, and Sequencing
For PCR, 200 ml of each sample in either Remel M5 or FMS
was used for DNA extraction and purification (QIamp DNA
Mini Kit, QIAGEN Sciences, Maryland) according to the man-
ufacturer’s instructions. Conventional PCR for mycoplasmas
was performed using primers to the 16S rRNA gene12 and
16S-23S intergenic spacer region (ISR).25 Amplified PCR
products were seperated in 2% agarose gels, stained with
ethidium bromide or GelRed (Biotium, Inc., Hayward,
California), and visualized with ultraviolet transillumination
for the expected DNA products. Selected 16S rRNA gene and
ISR amplicons were sequenced (Eton Bioscience Inc., Durham,
North Carolina; GENEWIZ, South Plainfield, New Jersey).
Amplicon forward and reverse sequences were visually
scanned for quality and miss-calls using the software FinchTV
(http://www.geospiza.com/Products/finchtv.shtml) to view the
chromatograms and select contiguous sequences. Contigs were
constructed and compared with 16S rRNA gene and ISR
sequences of Mycoplasma spp in GenBank using BlastN
(NCBI, USA) and ClustalW (biology workbench 2.2 software;
http://workbench.sdsc.edu).1,8
Results
Gross Pathological, Cytological, and Histopathological
Examination
All birds were in good postmortem preservation state. Vulture
Nos. 1 and 2 weighed 2.1 kg and were in good body condition
293
with adequate muscular mass and abdominal and subcutaneous
adipose tissue. Vulture No. 3 weighed 1.340 kg and was in a
markedly reduced body condition with moderate to marked atro-
phy of the pectoral musculature, no subcutaneous adipose tissue,
and a small amount of intracoelomic fat store. Both elbow joints
were moderately to markedly enlarged, and the soft tissues sur-
rounding them were firmly adherent to the skin (vulture Nos. 1,
2, and 3). In vulture No. 1, the elbow joints had increased mobi-
lity in the ventrodorsal plane, reduced range of motion upon
extension, and slight crepitus in the left elbow. The range of
motion of the elbows in vulture Nos. 2 and 3 was within normal
limits. Bilaterally, the joint capsules were distended and mark-
edly thickened, up to 4 mm thick. In the most severely affected
elbows, a large synovial cyst (up to 20  25  10 mm) commu-
nicating with the joint space and extending along the humerus in
between the extensor and flexor tendons and muscles was pres-
ent (vulture Nos. 1, 2 and 3). Articular spaces were filled with a
moderate to large amount of thick pink-white (vulture No. 1) or
translucid (vulture No. 3) viscous fluid containing a large
amount of 1- to 3-mm off-white caseous debris or thick dark
yellow-orange viscous fluid (vulture No. 2). The volume of joint
fluid exudate varied from 1 to 3 ml. The elbow articular surface
lesions ranged from a single focal 3-mm-diameter ulcer of the
distal humerus (vulture No. 2) or proximal ulna (vulture No.
3) to almost complete eburnation and/or replacement by firm
white tissue of the humeral, radial, and ulnar articular surfaces
(vulture No. 1) (Figs. 1, 2). In vulture Nos. 2 and 3, bilaterally,
a 20- to 40-mm long segment of gastrocnemius tendon overlying
and proximal to the hock joint was markedly thickened and firm.
The joint capsules of both hock joints (vulture Nos. 2 and 3) and
right elbow and carpus (vulture No. 3) were moderately thick-
ened, up to 2 mm thick, off-white, firm, and distended by a large
amount of thick viscous fluid similar to the fluid present in the
elbow joints. In vulture No. 3, the right metacarpophalangeal
joint was moderately enlarged and very firm and had restricted
range of motion. No joint space was present, and the articular
surfaces were replaced by firm and gritty white tissue that
bridged between the distal metacarpus and proximal phalange
(ankylosis). All other joints examined were within normal limits
including shoulders (vulture Nos. 1 and 2 only), carpal (vulture
Nos. 1 and 2 only), hips, stifles, hocks (vulture No. 1 only),
metatarsophalangeal joints, interphalangeal joints, cervical ver-
tebral joints, and thoracic vertebral joint.
Joint fluid cytology from affected joints revealed a severe
heterophilic inflammation. The smears were highly cellular
with a granular proteinaceous fluid background and little wind-
rowing of the cells, and, in vulture No. 1, were hemodiluted.
The cellular population was composed of mostly mature viable
heterophils, fewer degenerated heterophils, and mononuclear
cells admixed with a moderate amount of cell debris. Large
mononuclear cells often contained phagocytosed cell debris
and degenerated heterophils. The joint fluid white blood cell
number estimates ranged from 42,000 to 55,000 leucocytes/
ml. The 100 cell differential counts varied from 59% to 79%
heterophils, 12% to 20% large mononuclear cells, and 9% to
21% small mononuclear cells.
294 Veterinary Pathology 50(2)

Figure 1. Elbow; black vulture No. 1. (a) Normal elbow articular surfaces of an unaffected black vulture. (b) Severe chronic prolifera-
tive arthritis, left elbow. Marked joint capsule thickening, cartilage erosion, ulceration, and proliferation of granulation tissue (pannus)
are present. Figure 2. Radius and ulna; black vulture No. 2. Median section of formalin-fixed proximal radius and ulna. (a) Normal
radius and ulna from an unaffected black vulture. Note the thin regular layer of articular cartilage. (b) Severe chronic arthritis, left
elbow (vulture No. 2). Cartilage fibrillation (arrow), ulceration (arrow head) and thickening of subchondral bone plate are observed.
Wettere et al 295

Histopathological examination revealed a diffuse chronic
lymphoplasmacytic arthritis and tenosynovitis in the affected
joints (Fig. 5). The severity of the lesions varied from mild (right
elbow joint and hock joint of vulture No. 2) to moderate (left
elbow joint of vulture No. 2 and both elbows and right hock of vul-
ture No. 3) to severe (left and right elbow joints of vulture No. 1,
gastrocnemius tendons of vulture Nos. 2 and 3, and left hock joint
of vulture No. 3). In the less severely affected joints, the lesion
ranged from a mild to moderate lymphoplasmacytic infiltration
and fibrosis of the subsynovial connective tissue, mild synovium
hyperplasia, and no cartilage lesions, to diffuse fibrillation and
focal or multifocal ulceration and necrosis of the articular carti-
lage, synovium, and menisci (both hock joints and elbows,
respectively, in vulture Nos. 2 and 3) (Fig. 3). In the most severely
affected joints, there was almost complete ulceration of the articu-
lar surfaces with focal or multifocal loss of the subchondral bone
plate (right and left elbow of vulture No. 1; Fig. 4). An inflamed
layer of granulation tissue (pannus) covered the exposed subchon-
dral bone plate and extended in between epiphyseal bony trabecu-
lae as well as between the radius and ulna. The synovium was
diffusely ulcerated with only rare individualized hypertrophied
synoviocytes present. Multifocally variable amounts of luminal
cell debris, degenerated heterophils, and fibrin clots were
adherent to the subsynovial layer. The pannus tissue and the sub-
synovial connective tissue layer were infiltrated by numerous
plasma cells and fewer lymphocyte, macrophages, and hetero-
phils. Occasional lymphoid aggregates consisting of predomi-
nantly lymphocytes with peripheral plasma cells were present
in the subsynovium. Few scattered granuloma characterized by
a thin outer rim of multinucleated giant cells surrounding homo-
genous eosinophilic material with little karyorrhectic debris were
present in the subsynovial tissue, pannus, and bone epiphysis. A
subchondral synovial cyst lined by inflamed fibrovascular tissue
and communicating with the articular surface was present in the
left distal humerus (vulture Nos. 1 and 2) and in between the left
proximal radius and ulna (vulture No. 1). Multifocally, deposition
of woven bone was evident in the subchondral region of the epi-
physis (sclerosis) and along the epiphyseal periosteum (osteo-
phytes formation). In vulture Nos. 2 and 3, the gastrocnemius
tendon sheaths overlying the hock joints were markedly thick-
ened by fibrous tissue and an inflammatory cell infiltrate similar
to the one described in the joint synovium. A few scattered gran-
ulomas were also present. The tendon sheath synovium was ulcer-
ated with multifocal fibrinocellular clots adherent to the
subsynovial layer. In vulture No. 2, the gastrocnemius tendon was
multifocally hypercellular.
In addition, a chronic moderate focal lymphoplasmacytic
and hyperplastic ventriculitis with bacteria, fungal spores, and
pseudo-hyphae within the thickened koilin layer, and a severe
diffuse splenic amyloidosis were observed in vulture No. 2.
Moderate myeloid hyperplasia with immature heterophils rep-
resenting 75% of the bone marrow cell population was present
in all vultures. No significant histological lesions were
observed in the other organs examined.
Toxicology
The lead concentrations in the liver samples from vulture Nos.
1 and 3 were 0.08 and 0.4 mg/kg, respectively. Blood lead level
for vulture No. 2 was 7.9 mg/dl. No blood or liver tissue refer-
ence ranges for black vultures were available, but the lead lev-
els detected in these samples were consistent with normal
ranges for other avian species.24
Bacteriology
Routine aerobic (vulture Nos. 1, 2, and 3) and anaerobic (vul-
ture Nos. 2 and 3) bacterial cultures from heart blood, liver, and
joints did not grow any bacteria on plated media after 48 hours
of incubation and in broth culture after 7 days. There was some
evidence of mycoplasmal growth in first passage broth and agar
media with samples from both vulture Nos. 1 and 2. However,
growth was not sustained and could not be expanded or further
passaged. In vulture No. 3, mycoplasma organisms were iso-
lated from samples of the right carpus and left shoulder, pro-
ducing an acid shift color change in broth and typical
mycoplasma colonies on agar FMS media.
Mycoplasmas PCR and Sequencing
For vulture No. 1, 16S rRNA gene primers yielded positive
results with samples tested directly from the right elbow (joint
aspirate and 1 of 2 swabs). No amplicon was detected in the
sample from the left elbow joint. For vulture No. 2, PCR of the
left gastrocnemius tendon sheath sample was weakly positive;
results were negative from the right elbow joint, trachea, air
sac, and left hock joints on direct test. However, following
mycoplasma culture enrichment, a swab sample from the left
elbow joint was weakly positive. For vulture No. 3, PCR was
positive with samples from the left elbow and shoulder, both
hock joints, right carpus, and trachea. Selected amplicons from
vulture Nos. 1 and 3 (left shoulder, right carpus, and myco-
plasma isolate from the right carpus) were sequenced and
yielded 769, 876, 654, and 886 base pair products with
99.3%, 99.9%, 99.7%, and 99.9% match with the 16S rRNA
gene sequence of M. corogypsi (GenBank accession number
NR_025896), respectively. In vulture No. 2, the weak PCR

(Figure continued). Figure 3. Left hock joint; black vulture No. 3. Median section of formalin-fixed hock joint. (a) Severe necrosis of
the menisci (arrow) and thinning and blurring of the articular cartilage outline (arrowhead). The gastrocnemius tendon sheath is thick-
ened and contains inflammatory exudate (asterisk). (b) Hock joint histology section correlate. Severe diffuse fibrillation and necrosis of
the articular cartilage (arrow head) and necrosis of the meniscus (arrow), T, tibiotarsus; TM, tarsometatarsus; M, meniscus. Hematox-
ylin and eosin (HE). Figure 4. Proximal radius; black vulture No. 1. Severe ulceration of the cartilage and subchondral bone (arrow
head), proliferation of inflamed granulation tissue (short arrow and insert), and new trabecular bone formation (long arrow). Fibrino-
cellular debris partially covers the ulcerated articular surface. HE. Figure 5. Gastrocnemius tendon sheath; black vulture No. 2.
Severe plasmacytic tenosynovitis. HE.
296
amplicon was of poor quality and low concentration, and
sequencing resulted in a 284 base pair product with a 96%
match to the 16S rRNA gene sequence of M. corogypsi, due
to a small number of inconclusive base ‘‘N’’ reads throughout
the sequence. Direct sequencing of the PCR amplicon from the
trachea was not productive.
Amplification with ISR primers of both mycoplasma
isolates yielded the entire 302 base pair sequence of the
16S-23S ISR with 100% homology to the M. corogypsi ISR
sequence (GenBank accession number AJ780989.1).The docu-
mented polymorphic sites in the 16S-23S rRNA ISR sequence
of M. corogypsi at positions 1, 5, 64, and 120 were C, G, T, and
A, respectively, in our isolates.
Discussion
In all vultures, the lesions of diffuse chronic lymphoplasmacytic
polyarthritis with a fibrinoheterophilic joint exudate supported a
bacterial or viral cause or, less likely, an immune-mediated dis-
ease. No walled bacteria were isolated by routine bacteriology or
observed histologically. However, mycoplasmas were detected
by PCR from joints in all 3 vultures, and Mycoplasma spp was
isolated in 1 vulture (vulture No. 3). The amplicon sequences
identified M. corogypsi in vulture Nos. 1 and 3 and were sugges-
tive in vulture No. 2. Species identification was confirmed by
amplification and sequencing of the 16S-23S ISR of 2 myco-
plasma isolates. Restriction of the lesions to the articulations and
tendon sheaths, lack of detectable walled bacteria, and the
histological changes consisting of synovial cell proliferation,
ulceration, villus formation, and predominantly lymphoplasma-
cytic inflammatory infiltrate with formation of lymphoid
nodules were supportive of a mycoplasmal origin.6,26 Although
no walled bacteria were isolated by routine bacteriological
culture or seen histologically, it cannot be entirely ruled out that
the arthritis was caused by walled bacteria or a mixed infection
with walled bacteria and mycoplasma. The antibiotic treatment
administered in vulture Nos. 1 and 2 could have eliminated the
walled bacteria or impaired bacterial and mycoplasmal isolation
despite the termination of antibiotic therapy 11 days prior to
euthanasia and necropsy. An underlying viral cause such as a
reovirus infection also cannot be ruled out since histological
changes induced by reovirus are similar to Mycoplasma spp
infection, at least in poultry.6
The clinical presentation, gross necropsy findings, absence
of detectable walled bacterial infection, and presence of
mycoplasma nucleic acid within the affected joints are similar
among the 3 cases presented here and the previously published
case of polyarthritis in a black vulture.25 Identification and
isolation of M. corogypsi from the lesions in this report further
support M. corogypsi as the likely cause. Demonstration of the
presence of M. corogypsi within the articular lesions by immu-
nohistochemistry or in situ hybridization would be useful to
strengthen this association and study the pathogenesis of the
disease. Development and validation of an antibody or DNA/
RNA probes for M. corogypsi were beyond the scope of this
report.
Veterinary Pathology 50(2)
In Ruder et al,26 histology of the joint revealed an acute
fibrinoheterophilic arthritis lesion that suggested a bacterial
or an acute mycoplasmal infection. The characteristic morpho-
logical changes associated with chronic mycoplasmal arthritis
in poultry were absent. In these 3 vultures, possibly due to the
prolonged clinical history, the typical histological lesions of M.
synoviae or M. gallisepticum–induced chronic arthritis and
tenosynovitis were observed, thus corroborating the microbiol-
ogy and molecular test results.4,6
In all 3 vultures in this report, both elbows joints were
affected. Hock joints were affected in 2 of the 3 vultures as well
as in the case reported by Ruder et al.26 A carpus was affected
in 1 vulture and the case reported by Ruder et al.26 A shoulder
was also affected in 1 vulture. From these 4 cases, it seems that
there may be a predilection for the elbow and hock joints,
although all appendicular joints can be affected.
In contrast to Ruder et al,25 we observed no histological
changes in the respiratory system of the vultures in this study.
Bronchitis and/or lymphoid hyperplasia of the bronchus-
associated lymphoid tissue can be observed histologically with
Mycoplasma spp infection in poultry and would have provided
corroborating evidence of a mycoplasmal infection.6,25,28 The
presence of Mycoplasma spp in the respiratory system was
investigated by culture and mycoplasma PCR in vulture Nos.
2 and 3. An amplicon was obtained from a tracheal swab in
vulture No. 3, but sequencing of this amplicon failed. Thus,
detection of a Mycoplasma spp in the trachea could not be
confirmed.
Splenic amyloidosis was present in 1 vulture (vulture No. 2)
as in Ruder et al.26 The occurrence of splenic amyloidosis is not
surprising, as reactive systemic amyloidosis is not rare in birds
and often occurs as a consequence of chronic inflammation and
increased serum amyloid A protein.7
Ruder et al26 speculated that lead poisoning and presumed
immune suppression may have predisposed the vulture to
infection with M. corogypsi. The liver (vulture Nos. 1 and 3)
or blood (vulture No. 2) lead level detected in the 3 vultures
that we examined were low, less than 4 ppm wet weight and
0.2 mg/L, respectively, thus indicating that lead intoxication
was not a predisposing factor in these 3 vultures.24 No potential
predisposing factor was identified in vulture Nos. 1 and 2. In
vulture No. 3, the ankylosed left carpal joint was indicative
of an old chronic injury that likely had impaired flight
capability and may have weakened the bird and thus been a
predisposing factor.
Investigations of Mycoplasma spp in the respiratory system
of healthy birds of prey have revealed a high prevalence, rang-
ing from 47% to 100%.13,14,17 Such a high prevalence in appar-
ently healthy birds suggests that the Mycoplasma spp detected
are likely commensal organisms in the respiratory system of
raptors. Consequently, detection of Mycoplasma spp in a
respiratory lesion of a diseased raptor must be interpreted care-
fully. M. corogypsi has not been identified in the respiratory
tract in European raptor surveys using immunobinding and
PCR assays but has been detected in asymptomatic falcons in
the Middle East: 1 peregrine falcon (Falco peregrinus), 4 saker
Wettere et al
falcons, and 1 gyrfalcon  saker hybrid (Falco rusticolus
 cherrug).13,14,17 However, identification of M. corogypsi in
these falcons relied only on an immunobinding assay, and
therefore false-positive results cannot be excluded. The
Mycoplasma spp amplicon in the trachea of vulture No. 3 could
not be confirmed, unfortunately, and may have been spurious.
Epidemiological studies to determine the prevalence of
M. corogypsi in the respiratory tract and joints of New World
vultures would help clarify its possible role in polyarthritis and
tenosynovitis. Attempts at detection of mycoplasma 16S rRNA
gene from the joint fluid of the 2 elbows of a black vulture lack-
ing clinical, gross, and histological evidence of joint disease
using a mycoplasmas PCR was negative (A.V.W., D.L.,
L.D., unpublished data). However, confirmation that infection
with M. corogypsi causes polyarthritis in black vultures would
require experimental infection to fulfill Koch’s postulates.
This report emphasizes that veterinary clinicians and
pathologists should consider Mycoplasma spp as potential
pathogens in American black vultures and should obtain joint
fluid or tissue specimens for culture and molecular diagnosis.
Acknowledgements
The authors thank P. Jay, S. Horton, N. Whitehurst, M. Mattmuller, S.
Hyuan, M. Engelmann, C. Orlando, R. De Voe, J.B. Minter, B. Long,
V. Grunkemeyer, J. Benito, and the volunteers at the Carolina Raptor
Center and Valerie H. Schindler Wildlife Rehabilitation Center for
their technical and clinical assistance.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to
the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for
the research, authorship and/or publication of this article: A. J. Van
Wettere is supported by Ruth L. Kirschstein National Research Ser-
vice Award T32 RR024394 as part of NCSU’s Comparative Medicine
and Translational Research training program.
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