Abstract
Chlamydiosis is a global, zoonotic, bacterial disease caused by a heterogenous group of obligate intracellular, gram-negative bacteria with various clinical manifestations in birds and mammals, including humans. It has been reported in several species of wild birds with suspected or documented transmission to pet birds, captive raptors (falconer owned), dogs, cats, and humans.2-4 Relatively little is known about the epidemiology and pathogenesis in wild bird populations, partly because mortality events rarely exceed predator or scavenger removal. Consequently, little is known about the implications of wild bird infections to wildlife populations, domestic animals, and humans.
From November 2002 to March 2003, there was a cluster event of chlamydiosis in red-tailed hawks (RTHs, Buteo jamaicensis) in the vicinity of Davis, CA, affecting seven of the 20 RTHs submitted for necropsy to the Anatomic Pathology Service, Veterinary Medical Teaching Hospital (VMTH), Davis, CA. All affected RTHs were juveniles, in poor body condition, and with variable concurrent disease processes. Cell culture and molecular characterization of the isolate from one RTH suggested it was a new strain of Chlamydiophila psittaci, most closely related to strain M56. Trauma with subsequent debilitation is the most common presentation of RTHs to the VMTH for treatment and rehabilitation,8 and review of archived VMTH pathology cases over the last 20 years yielded only three cases of chlamydiosis out of 215 RTH necropsies. Thus, either this was an unusual morbidity/mortality event, possibly related to a new strain of C. psittaci, or chlamydiosis has been under-diagnosed.
Under the current taxonomic classification, the family Chlamydiaceae is subdivided into two genera: Chlamydia, which incorporates three species; and the newly designated Chlamydiophila, which incorporates six species including psittaci.1 Assignment to species is based on less than 0.8% difference in the 16S rRNA segment of genome. Within C. psittaci, eight strains are recognized based on monoclonal antibody tests, the polymerase chain reaction (PCR) of the 16S rRNA sequence, and PCR sequence analysis of the major outer membrane protein (MOMP). Six of the strains (A through F) are considered endemic in birds, including most pet birds, domestic fowl and poultry, and wild birds.1,2 In wild birds, however, too few isolates have been typed to know whether additional strains exist. The two remaining strains (WC and M56) have been isolated each once during mammalian epizootics, but are believed to have crossed from an avian host.10,11 Transmission may be horizontal or, infrequently, vertical, and may occur from live birds and fresh and chilled carcasses.2 The rate and success of transmission and pathogenicity is based on susceptibility of the host (with consideration of age, immune status, concurrent disease, and, possibly, genetic predisposition), the virulence of the strain for that host, the dose and route of infection, the persistence of infection and duration of shedding, and environmental factors.2,12
Diagnosis of avian chlamydiosis is relatively difficult, especially in antemortem cases. Antemortem confirmation requires a fourfold increase between acute and convalescent serum titers taken two weeks apart or positive culture from respiratory secretions. Trends in antibody production are variable among species and individuals. Some have undetectable antibodies, others never seroconvert with acute onset of disease, and others have persistent titers despite complete resolution of disease without further shedding.5-7 Consequently, it is difficult to correlate serum antibodies with active infection and potential for zoonotic transmission. Cell culture requires special handling procedures, is performed by only a few diagnostic laboratories due to zoonotic potential, technical demands and expense, and requires two to three weeks to ensure a negative result. Postmortem confirmation requires demonstration of the organism by cell culture, immunohistochemistry (IHC) or immunofluorescent antibody testing against the genus-specific LPS antigen of Chlamydia trachomatis, or a serum titer change with compatible lesions. Bacterial isolation in cell culture is still considered the gold standard in veterinary medicine because rapid, reliable tests available in human medicine, namely a PCR and antigen capture enzyme-linked immunosorbent assay (ELISA), have not been standardized in domestic and free-ranging species.2,7,9
This study was conducted (1) to determine whether other cases that occurred during the epizootic were caused by the new strain; (2) to determine the prevalence of chlamydiosis and this strain in RTHs in northern California; (3) to characterize gross, histologic, and immunohistochemical findings and thus identify associated lesions in RTHs; (4) to validate commercially available species-specific multiplex PCR (m-PCR, Gibco®) and genus-specific antigen-capture ELISA (Clearview™) and to develop a species- and strain-specific Taq-Man PCR (Lucy Whittier Molecular Core and Diagnostic Facility, Davis, CA). To address the first question, available samples from three additional RTHs from the epizootic were submitted for cell culture and strain identification. To determine prevalence, the sampling effort was increased to include not only all RTHs admitted to the VMTH, but also those admitted to the Lindsay Wildlife Hospital in the vicinity of San Francisco (Walnut Creek, CA). To characterize gross and histologic findings, a defined tissue set from each animal was submitted for routine histology and immunohistochemistry (IHC). To answer questions related to prevalence, diagnostic testing, and pathology, the ELISA, m-PCR, and species-specific Taq-Man PCR were run on fresh or frozen (-70°F) antemortem choana and cloacal swabs and postmortem liver, spleen, and air sac samples. The results were compared to findings from histology and IHC. Additionally, at necropsy liver, spleen, air sac, and coelomic swab were submitted for aerobic culture. Data was entered into the Epi Info™ program and statistical analysis run using chi-square, odds ratio, and sensitivity and specificity analyses.
To date, molecular testing confirmed that isolates from three additional cases from the cluster event had 100% homology with the index isolate. Consequently, this new strain of C. psittaci, designated strain G. Chlamydiosis, was diagnosed in 22.8% (13/57) of RTHs examined from November 2002 to April 2004, of which 11 cases occurred during the epizootic. Strain identification of the two cases from the non-epizootic RTHs is pending. All chlamydiosis cases were observed in juvenile birds (36.1%, 13/36). Infection was associated with season, as all cases occurred between December and April, constituting 48% of the cases submitted during this timeframe. There was no significant association found between Chlamydia infection and sex, chronic disease, or location; however, interpretation of the later variable is confounded by the relatively small sample size from the San Francisco area.
By gross, histologic, and immunohistochemical examination, several features had statistically significant association with chlamydiosis in juvenile RTHs. RTHs with chlamydiosis were 12.25 (95% CI, 1.6–138.48) times more likely to have hepatomegaly and 22.29 (95% CI, 2.63–988.14) times more likely to have hepatitis of increased severity. Hepatitis was defined as periportal to random, predominantly mononuclear inflammation. RTHs with chlamydiosis were also 7.2 (95% CI, 1.24–45.27) times more likely to have moderate to severe splenic hemosiderosis and increased renal mesangial proliferative glomerulopathy (chi square, p<0.001). Splenomegaly (27.8%, 10/36) with reticular sheath reticuloendothelial hyperplasia and airsacculitis (50%, 18/36) also were common lesions. A mild to moderate, mononuclear meningitis with predominantly intravascular and perivascular organisms (by immunohistochemistry) in the meninges and choroid plexus was identified in 23.1% (3/13) of the chlamydiosis cases. Finally, despite the lack of histologic lesions, the predominance of organisms identified by IHC in the ileocecal region, compared to low numbers in the colon, cloaca, and bursa, and few to none in the duodenum and jejunum, suggests that the ileocecal region is an excellent screening site for Chlamydia by IHC.
Thus far, there has been 100% concurrence between our gold standard test (histology supplemented by IHC) and Taq-Man PCR run on available antemortem choanal and cloacal swabs (n=33 total, n=2 Chlamydia birds; swabs were not available from the remaining 11 RTHs with chlamydiosis) and postmortem liver, spleen, and air sac samples (n=24 total, n=7 Chlamydia birds; tissue was not available from the remaining six RTHs with chlamydiosis). Comparing these results with those from ELISA and m-PCR, which were run on the same sample set, the data indicate that the latter two tests are equally specific but less sensitive, as there were four false-negative results. Regarding the ELISA, the absence of false-positive results despite the culture of gram-negative microbes from several cases, indicates that cross-reaction with the LPS antigen of other gram-negative microbes is not a real concern. Regarding the Taq-Man PCR, its sensitivity necessitates that molecular testing be interpreted in light of histology to accurately assess the contribution of chlamydiosis to morbidity and mortality of the bird. In summary, recommended samples to screen for chlamydiosis antemortem are pooled choana and cloacal swabs submitted for Taq-Man PCR. Recommended samples for postmortem diagnosis are pooled liver, spleen, and, potentially, ileum/ceca for Taq-Man PCR and histology supplemented by IHC.
Work currently is being performed to complete the testing and statistical analysis on the current tissue set. Also, a strain-specific Taq-Man PCR is being designed in order to identify the strain of C. psittaci that caused disease in the non-epizootic RTHs (2003–2004), as well as to provide a strain-specific, sensitive and specific diagnostic test for C. psittaci on ante- and postmortem specimens. Finally, continued ante- and postmortem sample submission for Taq-Man PCR will be used to increase the sample size to validate this test and to determine if molecular methods can replace cell culture as the gold standard for diagnosis of chlamydiosis.
Acknowledgments
The authors thank the Center for Companion Animal Health for funding this research, as well as Dr. Leslie W. Woods, Lydia L. Gan, Loren K. Jones, and the residents at the Companion Avian and Exotic Pet and Anatomic Pathology Services for their assistance.
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