Mycoplasma corogypsi associated polyarthritis and tenosynovitis in black vultures (Coragyps atratus)

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.

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 assumptions.^{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.

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 (meloxicam 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, Jacksonville, 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 discontinued except meloxicam until the bird was presented to the North Carolina State University College of Veterinary Medicine (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 anemia (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 Jersey) 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 during isoflurane anesthesia. The estimated WBC remained elevated 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.

Antibiotic therapy was discontinued, but analgesic therapy (meloxicam and tramadol [20 mg/kg every 12 hours orally, Amneal Pharmaceuticals, Hauppauge, New York]) was administered 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% heterophils, 34% lymphocytes, 2% eosinophils, and 8% monocytes). 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 recommended. Supportive care (fluids and feeding) and analgesic therapy (meloxicam) were administered until the bird was submitted 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 anesthesia 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% neutral 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. Sections 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 vulture 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 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 Scientific, 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 mycoplasma 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 manufacturer'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 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 atrophy 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 surrounding them were firmly adherent to the skin (vulture Nos. 1, 2, and 3). In vulture No. 1, the elbow joints had increased mobility 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 markedly thickened, up to 4 mm thick. In the most severely affected elbows, a large synovial cyst (up to 20 × 25 × 10 mm) communicating with the joint space and extending along the humerus in between the extensor and flexor tendons and muscles was present (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 thickened, 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 vertebral 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 windrowing 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.

Figure 1.

Elbow; black vulture No. 1. (a) Normal elbow articular surfaces of an unaffected black vulture. (b) Severe chronic proliferative 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.

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 vulture 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 cartilage, 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 articular 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 subchondral bone plate and extended in between epiphyseal bony trabeculae 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 subsynovial connective tissue layer were infiltrated by numerous plasma cells and fewer lymphocyte, macrophages, and heterophils. Occasional lymphoid aggregates consisting of predominantly lymphocytes with peripheral plasma cells were present in the subsynovium. Few scattered granuloma characterized by a thin outer rim of multinucleated giant cells surrounding homogenous 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 epiphysis (sclerosis) and along the epiphyseal periosteum (osteophytes formation). In vulture Nos. 2 and 3, the gastrocnemius tendon sheaths overlying the hock joints were markedly thickened by fibrous tissue and an inflammatory cell infiltrate similar to the one described in the joint synovium. A few scattered granulomas were also present. The tendon sheath synovium was ulcerated with multifocal fibrinocellular clots adherent to the subsynovial layer. In vulture No. 2, the gastrocnemius tendon was multifocally hypercellular.

Figure 5.

Gastrocnemius tendon sheath; black vulture No. 2. Severe plasmacytic tenosynovitis. HE.

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 thickened 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. Hematoxylin 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). Fibrinocellular debris partially covers the ulcerated articular surface. HE.

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 representing 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 reference ranges for black vultures were available, but the lead levels 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 (vulture 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 isolated from samples of the right carpus and left shoulder, producing 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 mycoplasma 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 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 documented 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 disease. 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 suggestive in vulture No. 2. Species identification was confirmed by amplification and sequencing of the 16S-23S ISR of 2 mycoplasma 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 lymphoplasmacytic 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 immunohistochemistry 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.

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 morphological 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 microbiology 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, ranging from 47% to 100%.^13,14,17 Such a high prevalence in apparently 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 carefully. 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 falcons, and 1 gyrfalcon x 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 lacking 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.