The Chlamydiaceae are one of the most successful groups of obligate intracellular, gram-negative bacteria and are responsible for a variety of diseases in animals and humans.1 The family Chlamydiaceae contains the single genus Chlamydia, and Chlamydia spp cause a variety of diseases affecting a wide range of species, including birds, reptiles, and mammals.1 Fourteen species are currently recognized within the genus, including C trachomatis, C pneumoniae, C pecorum, C abortus, C felis, C caviae, C muridarum, C suis, C psittaci, C avium, C gallinacea, C poikilothermis, C serpentis, and the recently identified C buteonis.2 There are also 3 candidate chlamydial species, including Candidate (Ca) ibidis, Ca C sanzinia, and Ca C corallus.2–7 Because of their wide host range, Chlamydia spp have significant impacts on human and animal health worldwide.8–10
One of the most widely distributed chlamydial species and frequently discussed zoonotic pathogens in avian populations is C psittaci. Chlamydia psittaci is globally distributed and has been reported in at least 469 domestic and free-living bird species, representing 30 orders.11–13 Like all Chlamydia species, C psittaci undergoes a biphasic life cycle between the infectious form—the elementary body—and the noninfectious, metabolically active form—the reticulate body.9 The elementary bodies are shed across mucous membranes in feces, urine, ocular and nasal discharges, and oropharyngeal fluids.9 The ability of C psittaci to infect a diversity of host cells, including epithelial cells and macrophages, leads to a wide range of clinical signs, which are well reported in psittacine birds.4,14,15 Clinical signs are variable and include conjunctivitis, ocular and nasal discharge, dyspnea, lethargy, diarrhea, anorexia, emaciation, and sudden death.4, 14,16 Transmission amongst birds primarily occurs via inhalation or ingestion of bacterial particles from dried feces, urine, and ocular or nasal secretions.4,10 Birds can shed infective particles as soon as 3 days following initial infection.9 The clinical course of disease and the duration of shedding varies between species and even amongst individuals within species.1
Bird-to-mammal transmission occurs through direct contact with infected birds, especially through inhalation of bird secretions or droppings.14,17 Chlamydia psittaci is environmentally stable and can remain infectious in dried feces for months.18
There are few reports of chlamydial infection causing clinical disease in free-ranging raptors in the US,19–22 but wild birds are considered an important source of transmission.10 In 1983, what was reported as C psittaci was isolated from 4 red-tailed hawks (Buteo jamaicensis; RTHAs) in Northern California, and in 1992, C psittaci was cultured from an RTHA in Louisiana with respiratory distress and diarrhea.19,20 A 2018 study22 reported a 1.37% prevalence of chlamydial DNA in healthy, free-ranging Buteo raptors in Northern California, although anti–C psittaci and anti–C pneumoniae antibodies were not identified in any of the birds using a commercially available elementary body agglutination (EBA) assay specific for these organisms. Among raptors undergoing rehabilitation in Oregon, 3.6% of raptors at centers were positive for C psittaci DNA on PCR.21 A 4.18% prevalence of chlamydial DNA in raptors in Central and Northern California was determined via PCR analysis of raptor swab specimens from 5 California wildlife rehabilitation centers.23 Chlamydial infections in wild birds are of concern because of the potential for zoonosis due to direct contact with wildlife rehabilitators, biologists, veterinarians, falconers, and the general public.10
Recently, a novel chlamydial species, C buteonis, was reported in free-ranging Buteo species, including RTHAs and a red-shouldered hawk (RSHA; B lineatus).2,22,23 Whole-genome sequencing performed on samples from the RSHA after the development of conjunctivitis and death revealed a novel chlamydial species phylogenetically intermediate to C psittici and C abortus, which was designated C buteonis.2 Since then, C buteonis has been confirmed via culture and whole-genome sequencing in an RTHA and a Swainson’s hawk (SWHA; B swainsonii) in Northern California, with 99.97% average nucleotide identity to the RSHA isolate.23 Anti–C buteonis immunoglobulin M antibodies were detected in RTHAs, SWHAs, Cooper’s hawks (Accipiter cooperii), and great horned owls (Bubo virginianus), indicating that the host range of C buteonis may extend beyond Buteo species.23 These findings suggest that C buteonis may be responsible for chlamydial disease previously attributed to C psittaci and C abortus in raptor species in Northern California. The host range is important to those working with wild birds in a conservation or rehabilitation setting as there are undocumented concerns that wild birds may transmit chlamydial infections to humans.24,25
The objectives of this retrospective study were to describe the prevalence, clinical findings, lesions, spatiotemporal distribution, and risk factors associated with chlamydial infections in free-ranging raptors presented to a university veterinary medical teaching hospital from 1993 through 2022.
Methods
Case and noncase selection
All electronic medical records were searched to identify free-ranging raptors admitted to the William R. Pritchard Veterinary Medical Teaching Hospital (VMTH), School of Veterinary Medicine, University of California-Davis (UC Davis), from January 1, 1993, through December 31, 2022. Medical records were initially reviewed to identify all raptor cases during this period, then refined to only include raptors in which the search term chlamy* was also found. The final search included only birds of the Buteo genus with the search term chlamy*. For this study, a bird was considered positive if it met the criteria for a confirmed or suspected case based on the guidelines proposed by the National Association of Public Health Veterinarians.16 These guidelines define a confirmed positive case as isolation via culture, Chlamydia spp DNA detection using in situ hybridization, or quantitative PCR (qPCR) of postmortem tissues in combination with characteristic pathology, clinical pathology, serology, or special staining (Gimenez or Machiavello). A suspected case was defined as one in which clinical signs were consistent with Chlamydia spp in combination with a positive qPCR result from mucosal swabs or tissue samples, serology, or epidemiologic evidence of a link to a confirmed case.16 Noncases were defined as wild raptors admitted to the UC Davis VMTH between the previously described dates that either were not tested for Chlamydia spp or tested negative for Chlamydia spp on all diagnostic tests. Birds were excluded from the study if the medical record was incomplete, if Chlamydia spp was suspected but no diagnostic testing was performed, or if diagnostic tests results were inconclusive.
Medical records review
For all animals included in this study, age class, sex, species, date of admission, and diagnostic test results were extracted from the medical record. Age class was based on plumage characteristics and divided into 3 categories: unknown, juvenile (hatch year and second year), and adult (third year and above). Sex was divided into 3 categories (male, female, and unknown) and was subjectively determined based on physical examination of species-specific sexually dimorphic body weight differences or on postmortem examination. A biochemistry panel and CBC were performed by the Clinical Pathology Service, UC Davis VMTH, for some patients; results were recorded if these diagnostic tests were performed. If a case had multiple CBC and biochemistry panels during hospitalization, the first panels performed closest to the date of admission were used for this analysis.
Pathology findings were recorded for all cases for which a necropsy was performed. Necropsies were performed by the Anatomic Pathology Service, UC Davis VMTH, within 24 to 48 hours of death or euthanasia. Tissues samples obtained from most patients included liver, spleen, air sacs, lung, heart, brain, thyroid and adrenal glands, proventriculus, ventriculus, colon, and small intestine for histologic examination. Samples were routinely fixed and processed for embedding, sectioned at 5 µm, and stained with H&E. Immunohistochemical (IHC) staining for Chlamydia spp was performed by the California Animal Health and Food Safety Laboratory from birds with suspicious histopathological lesions. Nonspecific IHC staining was evaluated with a duplicate slide receiving normal mouse whole immunoglobulin G in place of the Chlamydia-specific primary antibody. The stained slides were evaluated by board-certified veterinary pathologists (MKK, LJL, and TSZ).
Real-time quantitative mucosal PCR, quantitative tissue PCR, and genotyping
Mucosal swab samples were collected from suspect cases and stored at −20 °C for 24 to 48 hours until extraction. Mucosal qPCR from combination conjunctival, cloacal, and choanal swab samples was performed by the Real-Time PCR Research and Diagnostics Core Facility, UC Davis School of Veterinary Medicine, using standard laboratory procedures for targeting and amplifying the ompA genes of C felis and C psittaci.
Previously paraffinized tissue samples from Chlamydia-positive, Chlamydia-suspected and Chlamydia-negative raptors (n = 67) were retrieved from the UC Davis VMTH Anatomic Pathology Service, sectioned into 25-µm scrolls, and submitted to the University of Georgia Infectious Disease Laboratory for tissue PCR (tPCR) using commercial laboratory procedures.26
Twenty mucosal PCR (mPCR)-positive products were genotyped. Geneious Prime version 2023.0 (Dotmatics) was used to align strains of C abortus, C buteonis, C felis, and C psittaci to determine a location with mutations to distinguish between the different strains of the Chlamydia ompA gene. Three positive controls were used during the PCR and sequencing to ensure accuracy, including C psittaci from the National Veterinary Services Laboratory and plasmid DNA for C felis and C buteonis. Advantage 2 Polymerase (Takara Bio) was used for PCR according to the manufacturer’s instructions with the following cycling conditions: 4 minutes at 95 °C followed by 40 cycles of 1 minute at 95 °C, 1 minute at 52 °C, 1 minute at 72 °C, and a hold of 10 minutes at 72 °C to generate a 200-bp piece. The PCR product was resolved on 1.5% agar gel for amplicon confirmation and gel purification using the QIAquick Gel Extraction Kit (Qiagen) according to the manufacturer’s instructions. Sequencing was performed at the DNA Sequencing Facility, University of California-Berkeley. Geneious Prime (Dotmatics) was used to align the results for strain determination.
Statistical analysis
All statistical analyses were performed using R, version 4.2.2 (R Core Development Team). The chi-square test of independence and multivariable regression models were used to analyze the association between Chlamydia spp infection status and several risk factors. Risk factors included in the analysis were age class (juvenile, adult, and unknown), sex (male, female, and unknown), and season of admission (winter [December through February], spring [March through May], summer [June through August], and fall [September through November]). Species were collapsed into 2 taxa groups (RTHA and non-RTHA). To evaluate risk factors for infection, only data collected from 2000 through 2022 was used due to the low number of cases represented in the dataset from 1994 through 2000. The strength of association between risk factors and Chlamydia spp infection status was assessed using adjusted ORs and 95% CIs. Possible confounding variables and interaction effects were evaluated in the model-building process. A variable was considered a confounder if there was a greater than 10% change between unadjusted and adjusted OR estimates for the other variables in the model. Akaike information criterion was used to select the final parsimonious model. The Hosmer-Lemeshow test was used to determine the goodness of fit of the logistic regression model.
Spatiotemporal clustering of wild raptors positive for Chlamydia spp with known location information was evaluated using a Bernoulli model (SatScan, version 10.0; Information Management Services Inc).27,28 The suggested default maximum spatial cluster size of 50% of the population at risk was used. For the temporal analysis, a time interval of 1 month was used. A significance level of P ≤ .05 was used for all statistical analyses.
Results
Signalment and prevalence
A total of 3,983 wild raptors were admitted to the VMTH from January 1, 1993, through December 31, 2022. Of those, 270 birds were initially tested for chlamydial infection status via mPCR; the majority were RTHAs (n = 179; Table 1). Based on initial inclusion criteria, 228 raptors were included in the study population, which included 82 females, 74 males, and 72 birds of undetermined sex. Approximately 43% of the birds in the study population (99 of 228) were admitted to the VMTH during the winter season (December, January, and February).
Species of free-ranging raptors tested for Chlamydia spp via quantitative PCR (qPCR) of mucosal swabs (n = 270).
| Species | qPCR only (negative) | qPCR only (positive) |
|---|---|---|
| American kestrel (Falco sparverius) | 4 | 0 |
| Barn owl (Tyto alba) | 6 | 0 |
| Cooper’s hawk (Accipiter cooperii) | 2 | 0 |
| Ferruginous hawk (Buteo regalis) | 1 | 1 |
| Golden eagle (Aquila chrysaetos) | 9 | 0 |
| Great horned owl (Bubo virginianus) | 11 | 0 |
| Harris hawk (Parabuteo unicinctus) | 1 | 0 |
| Merlin (Falco columbarius) | 2 | 0 |
| Northern harrier (Circus cyaneus) | 1 | 0 |
| Peregrine falcon (Falco peregrinus) | 2 | 0 |
| Northern pygmy owl (Glaucidium californicum) | 1 | 0 |
| Red-shouldered hawk (Buteo lineatus) | 21 | 0 |
| Red-tailed hawk (Buteo jamaicensis) | 109 | 70 |
| Rough legged hawk (Buteo lagopus) | 0 | 1 |
| Sharp-shinned hawk (Accipiter striatus) | 1 | 0 |
| Swainson’s hawk (Buteo swainsoni) | 16 | 2 |
| Turkey vulture (Cathartes aura) | 2 | 0 |
| Western screech owl (Megascops kennicottii) | 4 | 0 |
| White-tailed kite (Elanus leucurus) | 3 | 0 |
| Total birds tested | 196 | 74 |
mPCR = PCR of mucosal swab from conjunctiva, choana, and cloaca.
The prevalence of birds that met the criteria for suspected or confirmed cases from the total raptor population presented was 1.9% (74 of 3,983). All of these cases were Buteo spp. The majority were RTHAs (n = 70) followed by SWHA (n = 2), a rough-legged hawk (B lagopus; n = 1), and a ferruginous hawk (B regalis; n = 1). Among those classified as suspected or confirmed, there were 48 juveniles, 20 adults, and 6 birds for which age class was undetermined as well as 28 females, 27 males, and 19 birds of undetermined sex. A logistic regression model assessing risk factors for testing positive for Chlamydia spp revealed that the odds of testing positive were 2.52 times higher in juvenile birds compared to adults (OR, 2.52; 95% CI, 1.27 to 5.00). As compared to the summer months (June through August), the odds of testing positive for Chlamydia were 15.83 times higher in birds presenting during the winter season (December through February; n = 47; OR, 15.83; 95% CI, 3.98 to 62.93) and 8.88 times higher during the spring season (March through May; n = 20; OR, 8.88; 95% CI, 2.03 to 38.79). The odds of testing positive for Chlamydia did not differ with sex of the bird.
Physical examination/comorbidities
Physical examinations were performed on the 74 cases. Physical examination findings were nonspecific; 74 of 74 (100%) were in poor body condition (body condition score, 1 to 2/9) and had some degree of dehydration (> 5%) at presentation. Most birds (68 of 74) were obtunded on presentation. Comorbidities were commonly observed among the confirmed and suspect cases, with all but 9 of 74 cases (12%) reported to have at least 1 comorbidity upon physical examination. Comorbidities ranged in severity from incidental (ectoparasitism) to severe (systemic aspergillosis) and were identified both antemortem and postmortem. Aspergillosis (systemic or localized to the lungs or air sacs) was present in 16 of 74 positive or suspected cases (22%). Ectoparasitism in the form of lice was most commonly reported in 12 of 74 cases (16%), and endoparasitism (including nematodiasis, trematodiasis, capillariasis, and coccidiosis) was reported in 29 of 74 cases (39%). Comorbidities associated with noninfectious diseases included traumatic injuries, such as fractures and traumatic brain injuries in 15 of 74 (20%) cases. Pododermatitis was also present in 6 of 74 cases (8%).
Complete blood count
Complete blood counts were performed on 53 of 74 cases (72%), and results are presented in Table 2. Seventy-five percent of cases had moderate-to-severe anemia (40 of 53) and leukocytosis (40 of 53).29 Mild-to-severe leukopenia also occurred in 12 of 53 cases (23%).29 More commonly identified abnormalities in 36 of 53 cases (68%) included moderate-to-severe heterophilia, with 35 of 53 having the presence of bands (66%) and 22 of 53 having monocytosis (42%).30,31 Thrombocyte numbers were considered to be adequate in most birds (45 of 53) but were only subjectively evaluated.
Complete blood count findings from raptors positive for Chlamydia spp (n = 53).
| Parameter | Mean ± SD | Median | Minimum | Maximum | Reference interval |
|---|---|---|---|---|---|
| RBC (X 103 cells/µL) | 2.07 ± 0.615 | 2.19 | 0.39 | 3.22 | 2.2–3.1 |
| Hct (%) | 28.43 ± 8.85 | 28.5 | 5 | 43 | 38–47 (PCV) |
| Leukocytes (X 103 cells/µL) | 36.16 ± 23.23 | 34.01 | 2.30 | 112.89 | 11–20 |
| Bands (X 103 cells/μL) | 6.08 ± 7.17 | 4.03 | 0.00 | 42.90 | |
| Heterophils (X 103 cells/µL) | 21.42 ± 16.91 | 17.12 | 0.00 | 78.80 | 4.8–13.2 |
| Lymphocytes (X 103 cells/µL) | 2.32 ± 6.22 | 0.95 | 0.00 | 44.36 | 2.1–10 |
| Monocytes (X 103 cells/µL) | 3.53 ± 3.85 | 2.38 | 0.00 | 16.28 | 0–0.6 |
| Eosinophils (X 103 cells/µL) | 1.35 ± 1.57 | 0.91 | 0.00 | 8.64 | 0–0.8 |
| Basophils (X 103 cells/µL) | 0.34 ± 0.46 | 0.15 | 0.00 | 1.68 | 0–0.4 |
| Total protein (g/dL) | 4.16 ± 1.77 | 4.2 | 0.04 | 7.5 | 3–5 |
| Fibrinogen (g/dL) | 372.48 ± 169.99 | 400 | 6.60 | 800 | 100–300 |
Biochemistry panel
Partial or complete biochemistry panels were performed on 35 of 74 cases (47%).31 Complete results are summarized in Table 3. Common findings included elevated creatinine kinase (CK) and AST and hypoalbuminemia. Mild-to-severe hypoglycemia was also identified in most evaluated birds. Elevated uric acid was commonly present and was most often associated with dehydration.
Biochemistry findings for Buteo raptors positive for Chlamydia spp infections (n = 35).
| Parameter | Mean ± SD | Median | Minimum | Maximum | Reference |
|---|---|---|---|---|---|
| Uric acid (mg/dL) | 14.75 ± 8.42 | 14.55 | 2.30 | 38.8 | 6–18 |
| Calcium (mg/dL) | 9.47 ± 1.58 | 9.45 | 6.6 | 12.3 | 8–13 |
| Phosphorus (mg/dL) | 4.97 ± 1.64 | 4.90 | 1.3 | 9.4 | 2–4 |
| Glucose (mg/dL) | 290.75 ± 97.75 | 285.00 | 56 | 628 | 222–388 |
| Total protein (g/dL) | 4.09 ± 1.90 | 4.05 | 0.4 | 8.4 | 3–5 |
| Albumin (g/dL) | 0.89 ± 0.38 | 0.90 | 0.2 | 1.5 | 0.9–1.2 |
| Globulin | 3.42 ± 1.57 | 3.30 | 0.2 | 7.2 | |
| AST (U/L) | 907.91 ± 2,539.18 | 429.50 | 199.00 | 15,650.00 | 160–495 |
| Creatinine kinase (U/L) | 8,987.14 ± 28,053.90 | 3,164.50 | 563 | 171,450.00 | 744–3,230 |
| Cholesterol (mg/dL) | 228.03 ± 454.93 | 145.00 | 17.00 | 2,796.00 | 132.64–269.9 |
| BUN (mg/dL) | 9.5 ± 8.37 | 7.00 | 1.00 | 37.00 | 9.33 |
| Glutamate dehydrogenase | 2.07 ± 3.74 | 0 | 0 | 16 |
Pathology/histopathology
Due to concerns of potential zoonosis, most positive birds that did not die in the hospital were euthanized and, in most cases, submitted for complete necropsy. Necropsies were performed on 58 of 74 cases (78%). Not all tissues were always collected at the time of necropsy; thus, some tissues were not available for evaluation. In 2 cases, limited organ samples were collected following euthanasia and were submitted for histopathology without a full necropsy being performed. Gross and microscopic lesions are summarized in Table 4. The spleen and liver were the organs most commonly and severely affected by lesions consistent with chlamydiosis followed by the air-sacs, lungs, kidneys, and heart. Mild-to-severe splenomegaly was noted in 25 of 53 examined samples (47%), and plasmacytic, histiocytic, lymphoplasmacytic, or granulomatous splenitis was noted in 22 of 53 samples (41%). The liver showed moderate-to-severe heterophilic, histiocytic, lymphocytic, granulomatous, or plasmacytic hepatitis in 44 of 56 cases (79%). Respiratory lesions included 21 of 43 fibrinous airsacculitis (48.4%), 9 of 43 granulomatous airsacculitis (20.9%), and 21 of 53 pneumonia (55.3%). Nephritis was the most common lesion of the kidney reported, along with renal tubular necrosis. The most common lesions in the heart were 21 of 39 histiocytic and lymphoplasmacytic myocarditis (53%), with 15 of 39 granulomatous to heterophilic pericarditis (38.5%) and 15 of 39 fibrinous epicarditis (38.5%).
Common histopathology findings from raptors positive with Chlamydia spp infection by organ.
| Organ | No. examined | Lesion | No. affected | Prevalence (%) |
|---|---|---|---|---|
| Liver | 56 | Hepatitis | 44 | 78.6 |
| Necrosis | 16 | 28.6 | ||
| Necrotizing hepatitis | 11 | 19.6 | ||
| Hemosiderosis | 10 | 17.9 | ||
| Extramedullary hematopoiesis | 9 | 16.1 | ||
| Kupffer cell hyperplasia | 8 | 14.3 | ||
| Hepatomegaly | 7 | 12.5 | ||
| Spleen | 53 | Splenomegaly | 25 | 47.2 |
| Splenitis | 22 | 41.5 | ||
| Extramedullary hematopoiesis | 17 | 32.1 | ||
| Lymphoid hyperplasia | 9 | 16.9 | ||
| Reticuloendothelial cell hyperplasia | 9 | 16.9 | ||
| Hemosiderosis | 9 | 16.9 | ||
| Necrosis | 9 | 16.9 | ||
| Air sac | 43 | Air sacculitis | 21 | 48.8 |
| Granulomatous airsacculitis | 9 | 20.9 | ||
| Fibrinous airsacculitis | 8 | 18.6 | ||
| Kidney | 39 | Nephritis | 24 | 61.5 |
| Interstitial nephritis | 14 | 35.9 | ||
| Tubular necrosis | 10 | 25.6 | ||
| Extramedullary hematopoiesis | 6 | 15.4 | ||
| Heart | 39 | Myocarditis | 21 | 53.8 |
| Pericarditis | 15 | 38.5 | ||
| Epicarditis | 15 | 38.5 | ||
| Lung | 38 | Pneumonia | 21 | 55.3 |
| Intralesional fungal hyphae | 10 | 26.3 | ||
| Necrotizing pneumonia | 8 | 21.1 | ||
| Hypoperfusion | 6 | 15.8 | ||
| Necrosis | 5 | 13.2 |
Intracellular bacteria consistent with Chlamydia spp were identified on chlamydial IHC in 40 of 45 cases evaluated (88.8%). One bird had positive IHC staining for intracellular bacteria in splenic, liver, air sac, kidney, and cardiac sections; many birds were IHC positive in more than 1 tissue.
Quantitative mPCR, tPCR, and genotyping
Quantitative mPCR and tPCR results are presented in Supplementary Table S1. Twenty-eight of the tissue samples tested positive for Chlamydia spp. Eighteen of these 28 tPCR results were positive using C buteonis–specific primers. Only 2 of the tPCR products were sequenced; both showed 100% homology with the C buteonis sequence. All 20 mPCR products submitted for genotyping were determined to be C buteonis.
Spatiotemporal analysis
One statistically significant cluster was noted in the spatiotemporal analysis. This cluster occurred from December 1, 2009, through December 31, 2012, and involved a total of 8 cases with a relative risk of 4.25. The cluster was centered at 38.568326 North, 121.611745 West over the Yolo Bypass with a radius of 13.85 km, encompassing the cities of Davis, CA, and Sacramento, CA (Figure 1).
Maps showing spatiotemporal distribution of raptors tested for Chlamydia spp infections and a cluster centered over the Yolo Bypass Wildlife Area, California.
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.09.0277
Discussion
This study determined that 74 of 270 raptor patients tested were positive for Chlamydia spp DNA, including C buteonis. Most studies22,23 evaluating chlamydial infections in raptors to date have found that the Buteo spp of raptors are primarily affected. But a recent study23 evaluating raptors presented to multiple rehabilitation centers throughout California found that several non-Buteo species tested positive for C buteonis via qPCR and antibodies as well. A separate genetic clade of C buteonis was also recently identified in 39 gyrfalcons (Falco rusticolus) from the United Arab Emirates, demonstrating new clades and that genotype classification of C buteonis is still in its infancy.32 Because many cases were tested before C buteonis–specific primers or sequencing were available, we cannot say with confidence that all cases are truly C buteonis.
Age class was significantly associated with Chlamydia infection status, with higher odds of juvenile hawks testing positive as compared to their adult counterparts. There are several factors that may explain this outcome. Juveniles may have a less robust immune system, which may predispose them to infection. Additionally, juveniles experience high mortality during their first winter as they are still learning to hunt. These birds are often emaciated and may be immunocompromised due to malnourishment and may be predisposed to multiple infections, including chlamydiosis. It is also possible that Chlamydia spp was potentially passed from asymptomatic parents to these chicks, and asymptomatic fledglings become symptomatic after becoming immunocompromised due to a lack of hunting skills.
The winter and spring months (December through May) were significantly associated with infection in this study. Seasonal patterns of infection with this pathogen are consistent with previously published work reporting the highest prevalence of chlamydial infections during the winter months.23 It is suspected that low temperatures and prey scarcity during the winter and early spring months leads to immunosuppression and increased susceptibility to infection and clinical disease in raptors, potentially explaining the seasonality observed in this study. In addition, it is possible that either a lower availability of prey or inefficient hunting skills for first-year birds results in increased competition and increased contact between wild bird species, thus increasing the risk of transmission.
Physical examination findings among Chlamydia-positive birds were nonspecific as is common among infected birds.16 Emaciation was a prevalent finding. This may be a result of chlamydiosis through factors such as illness-induced anorexia or decreased hunting ability. Alternatively, the emaciation could itself represent a risk factor for infection with Chlamydia spp. Unlike parrots affected with C psittaci, outward clinical signs of lower respiratory disease were not identified in this study. This is surprising given the high prevalence of lung and air sac lesions that was identified. Silent lower respiratory disease may have contributed to morbidity, allowing birds to be found and taken into captivity. Signs of upper respiratory disease, which is also commonly described among parrots with C psittaci, were also uncommon in this study.16
A wide range of abnormal hematological and biochemical values were identified in the infected birds in this study. However, hematology and biochemistry reference intervals for wild raptor species are scarce, and comorbidities make interpretation of these results challenging.30,33 Elevated total WBC count, anemia, heterophilia with left shift, and monocytosis were common laboratory findings among the infected birds in this study, and they are consistent with hematological findings in psittacine birds with chlamydiosis.16 Total protein was mildly elevated among cases, which may be reflective of poor hydration status, or it may be due to hyperglobulinemia as a result of infection. Elevated total protein has been described in psittacine birds with both acute and chronic inflammation.34 Furthermore, hyperglobulinemia has been reported in psittacine birds with chlamydiosis, which may represent another similarity between our study population and psittacine birds. In order to properly determine globulin concentrations, protein electrophoresis must be performed; unfortunately, this was not evaluated in this study. Lymphopenia was a common finding among the cases reported here. Lymphopenia is not consistent with other reported hematological findings of birds with chlamydiosis but is more commonly seen with viremia or stress.29,30
Biochemistry findings in our study included moderate elevations in AST, marked elevations in CK, and mild-to-severe hypoglycemia. Elevations in AST have also been described among parrots with chlamydiosis involving the liver.29 However, elevations in CK and AST together can indicate some degree of muscle damage and can occur secondary to trauma and emaciation, which were common presentations among this study population. The concurrent elevations in CK and AST are difficult to interpret, but given the pathology findings, it is likely that at least some of the elevations in AST can be attributed to hepatocellular damage. Unfortunately, bile acids were not evaluated in this study population. Hypoglycemia was prevalent among cases, but it is not commonly reported in other species with chlamydiosis. Common causes of hypoglycemia in avian species include prolonged starvation, septicemia, enterotoxemia, liver disease, and several endocrine disorders. Juvenile birds were more common in this study and are more susceptible to fluctuations in glucose than adults.30 While septicemia and liver disease were noted in some of the birds, further research is warranted to explore this finding.
The most common organs with documented lesions secondary to chlamydiosis in this study were the liver, spleen, air sacs, lungs, kidneys, and heart. Splenic changes, including splenomegaly, were common among the birds examined. Although splenomegaly is a nonspecific finding, chlamydiosis and mycobacteriosis are known to be common causes of splenomegaly among psittacine birds.35 Several cases in this study had airsacculitis, which is consistent with pathologic findings among infected psittacine birds, poultry, and pigeons.36 Cardiac lesions, including myocarditis, epicarditis, and pericarditis, were also common in our study. Cardiac lesions have been noted in RTHAs with West Nile virus and with highly pathogenic avian influenza.37,38 Cardiac lesions have been described in psittacine birds with septicemia secondary to chlamydiosis and in turkeys, pigeons, and chickens diagnosed with chlamydiosis.39 The presence of comorbidities in many of the birds of this study should be noted when interpreting the histopathology results.
Aspergillosis is commonly encountered in raptors with chlamydiosis because it is ubiquitous throughout the environment, and local or systemic infection in birds is often secondary to immunosuppression due to concurrent infection, stress, or malnutrition. It is possible that aspergillosis, in the cases reported here, was secondary to immunosuppression caused by primary chlamydial infection, or both aspergillosis and chlamydiosis could have been secondary to immunosuppression due to another cause. The high prevalence of trauma in our study population might be due to a predisposition of birds with chlamydiosis to injury as a result of decreased response times due to disease. The high prevalence of internal and external parasitism in our results (including pediculosis, gastrointestinal and air sac nematodiasis, trematodiasis, and coccidiosis) is probably incidental to chlamydiosis and is consistent with typical findings in wild birds.
Positive cases in this study were diagnosed through a variety of antemortem and postmortem diagnostic tests, including mPCR, tPCR, and IHC. Mucosal PCR from swabs collected from the conjunctiva, choana, and cloaca was performed on most birds on presentation or during hospitalization as this is one of the few available diagnostic tests that can be performed antemortem. Prior to the publication of the most recent guidelines for the diagnosis of C psittaci in birds in 2017, IHC was considered a confirmatory diagnostic test in parrots and was used as such in confirming chlamydiosis postmortem for many of the earlier birds in this study.16 The updated guidelines discuss concerns that commercial antibodies used in fluorescent antibody IHC staining frequently crossreact with nonepitopes, leading to false positive results. However, the board-certified pathologists for this study identified intracellular bacteria coinciding with positive IHC staining; most later cases had other confirmatory tests, such as mPCR and tPCR, along with IHC staining.
Serologic testing is notably absent from this data set. This is because the ELISA and immunofluorescent antibody (IFA) serological tests used prior to 2005 were not specific for avian chlamydial infections. The commercially available EBA test is specific for C psittaci, and the IFA test is only specific to the Chlamydia genus level. In a previous study,22 EBA and IFA were evaluated in 78 free-ranging RTHAs and SWHAs, and all tests were negative, whereas 4 of those birds tested positive via qPCR and genotyped via the ompA gene as “atypical” chlamydial organisms. The sequence from those 4 PCR products now directly aligns with C buteonis. A C buteonis–specific EBA was developed for experimental use, and, in a follow-up study evaluating sick and injured raptors in 5 rehabilitation centers in California, 12 EBA samples tested positive specifically for C buteonis.23
The spatiotemporal analyses revealed a significant spatiotemporal cluster centered over the Yolo Bypass Wildlife Area between the cities of Davis, CA, and Sacramento, CA. This is a natural area managed by the California Department of Fish and Wildlife and is part of the Pacific Flyway, a flyway for migratory birds that extends from Alaska to Patagonia. As a result, the area is highly concentrated with wild birds, including migratory bird species, including waterfowl and raptor species. Chlamydial organisms have been identified in wetland and migratory bird species, and it is possible that a larger outbreak of chlamydiosis has occurred among multiple wild bird species during this time frame in this area but was undetected.40 The spatial analysis was limited geographically by the catchment area of the VMTH as most birds were found in the surrounding area and presented to the VMTH by the general public. It is likely that sick or injured birds found outside of this region were admitted to other local wildlife rehabilitation organizations and are therefore not accounted for in this present study. This is a common limitation associated with studies involving clinical data.
The recognition and diagnosis of chlamydiosis in wild raptors is imperative to minimize the risk of potential but undocumented zoonotic transmission from infected raptors to exposed individuals, including veterinarians, veterinary technicians, and wildlife rehabilitators. Instances of zoonotic transmission from wild birds to humans are infrequent but should be taken seriously given the potential for severe disease in humans.25,41,42 The results of this study suggests that C buteonis is likely responsible for most chlamydial infections among wild Buteo species in California; however, it is not yet known whether this organism is zoonotic.22,23 Given that C buteonis has a close molecular relationship to C psittaci and C abortus, it is prudent to treat all C buteonis as potentially zoonotic. Season, clinical signs, and hematological findings may be useful in identifying suspect cases and implementing appropriate protections to prevent zoonotic transmission in clinical and wildlife rehabilitation settings. Further research is needed to understand the host range of this organism and the potential for transmission between raptors and other species.
There were limitations to this study. First, birds included in this study were admitted to a teaching hospital and, as such, were managed by students and house officers with varying degrees of familiarity with avian medicine. As a result, the detail and consistency of medical records varied, which may have influenced our results. Many of the birds included in this study suffered from 1 or more comorbidities, which may have altered the clinical and pathologic findings described. In addition, not all birds admitted to the VMTH were tested for Chlamydia spp, and there was variation in the types of diagnostic tests used over time. Furthermore, inferences about the epidemiology of this pathogen are limited as a result of bias toward clinically ill birds and individuals more easily detected in settings frequented by people and therefore may not accurately reflect the dynamics of this pathogen in the wild.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org.
Acknowledgments
The authors wish to thank the California Raptor Center volunteers, students, and staff that dedicated their time and effort into providing the daily care for these birds.
Disclosures
The authors have nothing to disclose. No AI-assisted technologies were used in the generation of this manuscript.
Funding
The authors have nothing to disclose.
References
- 2.↑
Laroucau K, Vorimore F, Aaziz R, et al. Chlamydia buteonis, a new Chlamydia species isolated from a red-shouldered hawk. Syst Appl Microbiol. 2019;42(5):125997. doi:10.1016/j.syapm.2019.06.002
- 3.
Bachmann NL, Fraser TA, Bertelli C, et al. Comparative genomics of koala, cattle and sheep strains of Chlamydia pecorum. BMC Genomics. 2014;15(1):667. doi:10.1186/1471-2164-15-667
- 4.↑
Sachse K, Bavoil PM, Kaltenboeck B, et al. Emendation of the family Chlamydiaceae: proposal of a single genus, Chlamydia, to include all currently recognized species. Syst Appl Microbiol. 2015;38(2):99–103. doi:10.1016/j.syapm.2014.12.004
- 5.
Staub E, Marti H, Biondi R, et al. Novel Chlamydia species isolated from snakes are temperature-sensitive and exhibit decreased susceptibility to azithromycin. Sci Rep. 2018;8(1):5660. doi:10.1038/s41598-018-23897-z
- 6.
Vorimore F, Hsia RC, Huot-Creasy H, et al. Isolation of a new Chlamydia species from the feral sacred ibis (Threskiornis aethiopicus): Chlamydia ibidis. PLoS One. 2013;8(9):e74823. doi:10.1371/journal.pone.0074823
- 7.↑
Taylor-Brown A, Ruegg S, Polkinghorne A, Borel N. Characterisation of Chlamydia pneumoniae and other novel chlamydial infections in captive snakes. Vet Microbiol. 2015;178(1–2):88–93. doi:10.1016/j.vetmic.2015.04.021
- 8.↑
Burnard D, Polkinghorne A. Chlamydial infections in wildlife-conservation threats and/or reservoirs of ‘spill-over’ infections? Vet Microbiol. 2016;196:78–84. doi:10.1016/j.vetmic.2016.10.018
- 9.↑
Wellehan JFX, Lierz M, Phalen DN, et al. Infectious disease In: Current therapy in avian medicine and surgery, Speer B ed. Elsevier, Maryland Heights, MO; 2016:22–106.
- 10.↑
Mamun T, Rahman J, Hossain M, Hasan R, Neoaj MT, Mia MM. Global prevalence of Chlamydia infection among wild birds as public health concerns: a systematic review and meta-analysis. Bulg J Vet Med.. 2023. doi:10.15547/bjvm.2023-0060
- 11.↑
Read TD, Joseph SJ, Didelot X, Liang B, Patel L, Dean D. Comparative analysis of Chlamydia psittaci genomes reveals the recent emergence of a pathogenic lineage with a broad host range. MBio. 2013;4(2):e00604-12. doi:10.1128/mBio.00604-12
- 12.
Wolff BJ, Morrison SS, Pesti D, et al. Chlamydia psittaci comparative genomics reveals intraspecies variations in the putative outer membrane and type III secretion system genes. Microbiology (Reading). 2015;161(7):1378–1391. doi:10.1099/mic.0.000097
- 13.↑
Kaleta EF, Taday EM. Avian host range of Chlamydophila spp. based on isolation, antigen detection and serology. Avian Pathol. 2003;32(5):435–461. doi:10.1080/03079450310001593613
- 14.↑
Harkinezhad T, Geens T, Vanrompay D. Chlamydophila psittaci infections in birds: a review with emphasis on zoonotic consequences. Vet Microbiol. 2009;135(1–2):68–77. doi:10.1016/j.vetmic.2008.09.046
- 15.↑
Knittler MR, Berndt A, Böcker S, et al. Chlamydia psittaci: new insights into genomic diversity, clinical pathology, host–pathogen interaction and anti-bacterial immunity. Int J Med Microbiol. 2014;304(7):877–893. doi:10.1016/j.ijmm.2014.06.010
- 16.↑
Balsamo G, Maxted AM, Midla JW, et al. Compendium of measures to control Chlamydia psittaci infection among humans (psittacosis) and pet birds (avian chlamydiosis), 2017. J Avian Med Surg. 2017;31(3):262–282. doi:10.1647/217-265
- 17.↑
Tolba HMN, Abou Elez RM, Elsohaby I. Risk factors associated with Chlamydia psittaci infections in psittacine birds and bird handlers. J Appl Microbiol. 2019;126(6):404–410. doi:10.1111/jam.14136
- 18.↑
Corsaro D, Venditti D. Emerging chlamydial infections. Crit Rev Microbiol. 2004;30(2):75–106. doi:10.1080/10408410490435106
- 19.↑
Fowler ME, Schulz T, Ardans A, Reynolds B, Behymer D. Chlamydiosis in captive raptors. Avian Dis. 1990;34(3):657–662. doi:10.2307/1591260
- 20.↑
Mirande LA, Howerth EW, Poston RP. Chlamydiosis in a red-tailed hawk (Buteo jamaicensis). J Wildl Dis. 1992;28(2):284–287. doi:10.7589/0090-3558-28.2.284
- 21.↑
Jouffroy SJ, Schlueter AH, Bildfell RJ, Rockey DD. Rhabdochlamydia spp. in an Oregon raptor. J Vet Diagn Invest. 2016;28:473–476. doi:10.1177/1040638716646408
- 22.↑
Lujan-Vega C, Hawkins MG, Johnson CK, et al. Atypical Chlamydiaceae in wild populations of hawks (Buteo spp.) in California. J Zoo Wildl Med. 2018;49(1):108–115. doi:10.1638/2017-0053R.1
- 23.↑
Seibert BA, Keel MK, Kelly TR, et al. Chlamydia buteonis in birds of prey presented to California wildlife rehabilitation facilities. PloS One. 2021;16(10):e0258500. doi:10.1371/journal.pone.0258500
- 24.↑
Marchino M, Rizzo F, Barzanti P, et al. Chlamydia species and related risk factors in poultry in north-western Italy: possible bird-to-human transmission for C. gallinacea. Int J Environ Res Pub Health. 2022;19(4):2174. doi:10.3390/ijerph19042174
- 25.↑
Kalmar ID, Dicxk V, Dossche L, Vanrompay D. Zoonotic infection with Chlamydia psittaci at an avian refuge centre. Vet J. 2014;199(2):300–302. doi:10.1016/j.tvjl.2013.10.034
- 26.↑
Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. P Natl Acad Sci USA. 1977;74(12):5463–5467. doi:10.1073/pnas.74.12.5463
- 27.↑
Kulldorff M, Nagarwalla N. Spatial disease clusters: detection and inference. Stat Med. 1995;14(8):799–810. doi:10.1002/sim.4780140809
- 28.↑
Kulldorff M, Athas WF, Feurer EJ, Miller BA, Key CR. Evaluating cluster alarms: a space-time scan statistic and brain cancer in Los Alamos, New Mexico. Am J Public Health. 1998;88(9):1377–1380. doi:10.2105/AJPH.88.9.1377
- 29.↑
Capitelli R, Crosta L. Overview of psittacine blood analysis and comparative retrospective study of clinical diagnosis, hematology and blood chemistry in selected psittacine species. Vet Clin Exot Anim Pract. 2013;16(1):71–120. doi:10.1016/j.cvex.2012.10.002
- 30.↑
Samour J. Diagnostic value of hematology. Clinical Avian Medicine. Vol 2. Harrison GJ, Lightfoot TL: Spix Publishing; 2015:587–640.
- 31.↑
Joseph V. Raptor hematology and chemistry evaluation. Vet Clin North Am Exot Anim Pract. 1999;2(3):689–699. doi:10.1016/s1094-9194(17)30116-0
- 32.↑
Vorimore F, Aaziz R, Al Qaysi L, et al. Detection of a novel genotype of Chlamydia buteonis in falcons from the Emirates. Vet Microbiol. 2024;291:110027. doi:10.1016/j.vetmic.2024.110027
- 33.↑
Black PA, McRuer DL, Horne LA. Hematologic parameters in raptor species in a rehabilitation setting before release. J Avian Med Surg. 2011;25(3):192–198. doi:10.1647/2010-024.1
- 34.↑
Rosenthal KL, Fudge AM. Avian protein disorders. Laboratory Medicine: Avian and Exotic Species. W. B. Saunders Co; 2000:171–173.
- 36.↑
Van Lent S, Piet JR, Beeckman D, et al. Full genome sequences of all nine Chlamydia psittaci genotype reference strains. J Bacteriol. 2012;194(24):6930–6931. doi:10.1128/JB.01828-12
- 37.↑
Ellis AE, Mead DG, Allison AB, Stalknecht DE, Howerth EW. Pathology and epiemiology of natural West Nile viral infection of raptors in Georgia. J Wildl Dis. 2007;43(2):214–223. doi:10.7589/0090-3558-43.2.214
- 38.↑
Shearn-Bochsler VI, Knowles S, Ip H. Lethal infection of wild raptors with highly pathogenic avian influenza H5N8 and H5N2 viruses in the USA, 2014–15. J Wildl Dis. 2019;55(1):164–168. doi:10.7589/2017-11-289
- 39.↑
Saif YM, Swayne DE, Pantin-Jackwood MJ, et al. Emerging diseases and diseases of complex or unknown etiology. In: Swayne DE, Boulianne M, Logue CM, et al, eds. Diseases of Poultry. 14th ed. 2020:1383–1410.
- 40.↑
Blomqvist M, Christerson L, Waldenstrom J, Herrmann B, Olsen B. Chlamydia psittaci in Swedish wetland birds: a risk to zoonotic infection? Avian Dis. 2012;56(4):737–740. doi:10.1637/10105-022812-ResNote.1
- 41.↑
Telfer BL, Moberley SA, Hort KP, et al. Probable psittacosis outbreak linked to wild birds. Emerg Infect Dis. 2005;11(3):391. doi:10.3201/eid1103.040601
- 42.↑
Rehn M, Ringberg H, Runehagen A, et al. Unusual increase of psittacosis in southern Sweden linked to wild bird exposure, January to April 2013. Euro Surveill. 2013;18(19):20478.
