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Evaluating 21-day doxycycline and azithromycin treatments for experimental Chlamydophila psittaci infection in cockatiels (Nymphicus hollandicus).

Abstract: To determine the efficacy of 21-day therapy with azithromycin and doxycycline in the treatment of experimental infection with Chlamydophila psittaci in cockatiels (Nymphicus hollandicus), 30 birds randomly assigned to 3 treatment groups and 1 control group were inoculated with Cpsittaci by combined intranasal and ocular routes. Morbidity, mortality, and results of polymerase chain reaction testing confirmed that infection was successful. Birds in group 1 (n = 8) received azithromycin at 40 mg/kg PO q48h for 21 days; in group 2 (n = 8), doxycycline at 35 mg/kg PO q24h for 21 days; in group 3 (n = 8), doxycycline at 35 mg/kg PO q24h for 45 days; and, in group 4 (controls; n = 6), no treatment. Six birds died either before or within 2 days of initiating treatment: 4 in the 3 treatment groups and 2 in the control group. Clinical signs resolved and mortality ceased 2 6 days after treatment was initiated in all treatment groups, whereas birds in the control group exhibited clinical signs for the duration of the study. Plasma doxycycline concentrations were measured during the treatment period and exceeded 1 [micro]g/mL at all time points. The absence of clinical signs and mortality in the treatment groups, even after inducing an immunocompromised state with dexamethasone (3 mg/kg IM q24h for 5 days), starting on day 70 postinoculation, suggested that treatment resulted in elimination of the pathogen. After euthanasia of the remaining 24 birds, 23 of the carcasses were submitted for necropsy. Spleen and liver samples from the birds in all treatment and control groups were polymerase chain reaction negative for Cpsittaci nucleic acid, and organisms were not detected by Gimenez stain. No gross or histologic differences were observed in the livers and spleens of treated and untreated infected birds. Lesions consistent with avian chlamydiosis (hystiocytosis) were seen in all birds and were considered residual. In this study, a 21-day course of either doxycycline or azithromycin was effective in eliminating C psittaci infection in experimentally inoculated cockatiels. Additional studies are necessary to evaluate the efficacy of these treatments in naturally infected cockatiels as well as other species of birds.

Key words: Chlamydophila psittaci, avian chlamydiosis, treatment, doxycycline, azithromycin, avian, cockatiel, Nymphicus hollandicus

Introduction

Avian chlamydiosis is caused by Chlamydophila psittaci, a gram-negative, obligate, intracellular bacteria. (1) The most recent list of susceptible birds includes 469 species that comprise 30 orders. (2) The order Psittaciformes contains species that are most susceptible to infection. (2) The clinical signs are variable and commonly involve the respiratory, gastrointestinal, nervous, and ocular systems. (3) The antemortem diagnosis of chlamydiosis requires a combination of diagnostic tests? The gross and histologic diagnosis of avian chlamydiosis requires demonstration of the pathogen by histochemical, immunohistochemical, or in situ hybridization techniques? Chlamydial infections can cause economic losses in both the pet bird and poultry industries, and represent a public health threat to people in close contact with infected birds. (6)

Chlamydiae are obligate intracellular organisms with a biphasic life cycle. Elementary bodies are inhaled or ingested, attach to the host cell membrane, and enter the cell and form an endocytoplasmic vesicle. The elementary body transforms into the reticulate body and replicates by binary fission. After division, the reticulate bodies transform into elementary bodies and are released, with or without lysis of the host cell. The whole cycle requires 48-72 hours, depending on infecting strain, host, and environmental conditions? However, the classic hypothesis of a lyc biphasic cycle seems to reflect only optimized growth conditions and could be extended by persistence as a third state. (7) Chlamydial persistence is known as a state of infection during which the pathogen remains viable but nonculturable, whereas the host immune system is incapable of eliminating it. (8) However, documentation of chlamydial persistence in natural infections remains controversial, and a mechanism for persistence has not been demonstrated in birds. Reliable markers (ie, genetic or protein) for documenting persistent chlamydial infection have not been developed. (7,9)

Doxycycline, a semisynthetic tetracycline, is considered the drug of choice for treating avian chlamydiosis. (10) This antibiotic inhibits protein synthesis by reversibly binding to 30S ribosomal subunits, (11) and it has numerous pharmacologic advantages compared with other tetracyclines. (12) Doxycycline has a higher oral bioavailability and a longer elimination half-life than other tetracyclines, which results in lower dosing regimens (improving palatability) and less-frequent dosing (improving compliance). (12) It is considered a bacteriostatic antibiotic and is presumed to be active only during the replication phase in the life cycle of chlamydiae. Because the organism is intracellular and unsubstantiated historic observations with marginally effective antimicrobials suggested posttreatment persistence of infection, prolonged treatment periods of 30-45 days have been recommended. (13,14) Plasma levels of 1 [micro]g/mL for the duration of the treatment period are thought to inhibit C psittaci replication? (12,15-20) Pharmacokinetic studies have been completed in several avian species. (21-25) Studies that evaluated plasma levels after parenteral and oral administration (16,26) and after treatment with medicated food and water have been reported. (15,17-20,27) In several psittacine bird species, once daily oral administration of doxycycline could maintain appropriate plasma levels (K. F., unpublished data, 1997), (12) and a dosage of 25-35 mg/kg PO q24h has been recommended for cockatiels (Nymphicus hollandicus). (10) Although these doses and regimens are generally accepted for psittacine bird species, compliance is often problematic. Reducing the duration of treatment from the current recommendation (45 days) would likely increase compliance and ensure successful treatment of a potentially zoonotic pathogen.

Azithromycin, like other macrolide antibiotics, interferes with bacterial protein synthesis by binding to the 50S ribosomal subunit. (28) This antibiotic provides broad spectrum coverage against several potential pathogens, including gram-positive and gram-negative organisms, spirochetes, some anaerobic bacteria, and Chlamydophila species. (29) Azithromycin is generally considered a bacteriostatic antibiotic, although bactericidal activity has been demonstrated in vitro against C psittaci. (30) To date, only a single pharmacokinetic study has been done of azithromycin in an avian species, the blue and gold macaw (Ara ararauna). Based on plasma level results, the recommended dosage of azithromycin in this species was 10 mg/kg PO q48h for 5 treatments for nonintracellular bacterial infections and 40 mg/kg PO q24h for 30 days for intracellular bacterial infections. (29) The purpose of this randomized controlled clinical trial was to evaluate 21-day doxycycline and azythromycin treatments against experimental C psittaci infections in cockatiels. The hypothesis of the study was that a 21-day course of doxycycline or azithromycin would be as effective as a 45-day course of doxycycline in eliminating C psittaci infections in cockatiels, even after inducing an immunocompromised state in the cockatiels through corticosteroid treatment. Efficacy of the treatment was evaluated by the presence of clinical signs, gross and histologic lesions, and results of polymerase chain reaction (PCR) testing for C psittaci.

Materials and Methods

This study was performed in accordance with the regulations established by the Louisiana State University Animal Care and Use Committee. Thirty adult cockatiels of undetermined sex and age were used in this study. The cockatiels were obtained from a private aviary and were individually numbered with a wing band. All of the birds were examined and determined to be clinically normal, and 60 days were allowed for acclimatization. Whole blood and a combined choanal-cloacal swab sample from each bird was tested within 2 days after sampling for the presence of C psittaci nucleic acid by a PCR-based assay. Blood samples (0.7-1 mL) were collected from the right jugular vein with a 3-mL syringe and 26-gauge needle and transferred to dipotassium ethylenediaminetetraacetic acid tubes (Microtainer, Becton Dickinson and Company, Franklin Lakes, N J, USA). Whole blood and swab samples were submitted to the Emerging Diseases Research Group at the University of Georgia College of Veterinary Medicine, Athens, GA, USA, within 24 hours of sampling by overnight mail with frozen gel packs. For PCR testing, a pair of proprietary oligonucleotide primers derived from the highly conserved regions of the published ompl sequences for the MOMP (major outer membrane protein) gene of Cpsittaci were used. (31) A commercial real-time PCR-based test system was used to amplify and detect nucleic acid products.

The birds were randomly assigned to 3 treatment groups of 8 birds per group (groups 1-3) and 1 control group of 6 infected and non-treated birds (group 4) by using a random number generator. The birds were housed individually in cage racks in 3.0 x 4.6 m restricted-access laboratory animal rooms. Tap water and a commercial cockatiel seed mix (Kaytee Forti-Diet, Kaytee Products Inc, Chiton, WI, USA) were provided. Fluorescent lighting was maintained on an automatic timer with a 12-hour photoperiod.

The C psittaci organisms (serovar A, Louisiana State University isolate 84-1832) used to infect the birds were isolated from a cockatiel diagnosed with avian chlamydiosis by PCR testing and culture. The original challenge stock was low-passage cultured in the yolk sacs of embryonated hen's eggs. Aliquots of 1 mL of 20% infected yolk sac suspension in phosphate-buffered sucrose solution were stored at -70[degrees]C until required. A 1-mL aliquot of the culture was thawed and quantified by using the number of fluorescent foci (Chlamydophila cytoplasmic inclusion) in Vero cell tissue culture. (32) The inoculum was adjusted to 108 infected foci per 0.1 mL. A 1-mL tuberculin syringe fitted with a 20-gauge needle was used to place 0.05 mL of inoculum onto the corneal surface of the left eye, and a second aliquot of 0.05 mL was instilled into the right external naris.

The cockatiels were monitored daily for the presence of rhinitis, conjunctivitis, dyspnea, diarrhea, and depression. All the birds were tested for the presence of C psittaci nucleic acid at 16 days postinoculation (day 0) by PCR testing on samples of whole blood and a combined choanal-cloacal swab. At 16 days postinoculation (day 0), treatment groups (1-3) were placed in separate rooms from the positive control group (4), and treatment was initiated. Birds in group 1 received azithromycin (Zithromax oral suspension, 100 mg per 5 mL, Pfizer Pharmaceuticals, New York, NY, USA) at 40 mg/kg PO q48h for 21 days, group 2 received doxycycline (Vibramycin calcium, doxycycline suspension, 50 mg per 5 mL, Pfizer Pharmaceuticals) at 35 mg/kg PO q24h for 21 days, and group 3 received doxycycline at 35 mg/kg PO q24h for 45 days. Birds in group 4 (control) were not treated. All treatments were administered by a metallic feeding tube into the crop. On day 55 postinoculation, all the birds, including the positive control group, were tested for C psittaci nucleic acid by PCR testing of samples of whole blood and a combined choanal-cloacal swab.

On days 14 and 21 postinoculation, blood samples were collected from all 30 birds to measure doxycycline in plasma from the doxycycline-treated birds 2-4 hours after administration. Although plasma levels were measured only in the doxycycline treated birds in groups 2 and 3, all birds were sampled to minimize any bias introduced by handling and venipuncture. The blood samples were collected with a 3-mL preheparinized syringe and 26-gauge needle by the technique described previously. The blood was transferred to 3-mL plain tubes, centrifuged (10 000 g x 5 minutes), and decanted within 2 hours after collection. The plasma samples were stored at -70[degrees]C for later analysis. Plasma doxycycline concentrations were measured by reverse-phase high-performance liquid chromatography by using ultraviolet detection. (19) The assay was validated by measuring banked plasma samples and plasma spiked with known concentrations of doxycycline. The limit of quantification was 0.25 [micro]g/mL.

The birds of each treatment group, but not the positive control, received dexamethasone at 3 mg/ kg IM q24h for 5 days starting on day 70. The birds were monitored daily for rhinitis, conjunctivitis, dyspnea, diarrhea, and depression, and were tested for the presence of C psittaci nucleic acid by PCR testing of whole blood and combined choanal-cloacal swab samples collected on days 85 and 105. During the study, the birds with severe clinical signs were euthanatized by injection of pentobarbital sodium (0.1 mL per 100 g body weight; Beuthanasia-D, Schering-Plough Animal Health Corp, Union, N J, USA) into the subocciptal sinus. The surviving birds were euthanatized on day 105 by using the same method.

The surviving birds that were euthanatized were submitted for necropsy. Liver and spleen were obtained for histopathologic examination and PCR testing. Tissue samples for histopathologic examination were fixed in 10% neutral buffered formalin for 24 hours, embedded in paraffin blocks, cut into 5-[micro]m sections, and stained with hematoxylin and eosin and with Gimenez stains. The presence of C psittaci DNA was confirmed by PCR testing. The PCR technique used for testing liver and spleen samples differed from that used previously for whole blood and combined choanal-cloacal swab samples, and was processed at Louisiana State University. To assess the amplification, a 5[micro]L PCR reaction was subjected to electrophoresis on a 2% agarose gel stained with ethidium bromide and photographed under ultraviolet illumination.

A Fisher exact test (4 x 2 table) was used to determine if differences were present among the study groups when the outcome variable was dichotomous (eg, PCR test results among the groups). When differences were present, an additional exact test (2 x 2 table) was used to further elaborate the difference and to determine if sex had an effect on the outcome of the PCR results. The McNemar test was used to determine if the frequency of positive results differed between testing samples (eg, choanal-cloacal swabs versus blood samples). Data were analyzed by using a statistical software program (SPSS 11.0, SPSS Inc, Chicago, IL, USA) was used to analyze the data. Results were considered significant at P < .05.

Results

Chlamydophila psittaci nucleic acid was not detected in samples from any of the 30 cockatiels before inoculation. On day 16 postinoculation (day 0), choanal-cloacal swab samples from all birds were positive for C psittaci nucleic acid by PCR testing (Table 1). There was a significant difference in PCR test results in the birds 55 days after the initiation of treatment (P < .001). The difference identified posttreatment was attributed to comparisons made between the control group and the treatment groups. The control group was significantly more likely to be PCR positive than the birds treated with azithromycin for 21 days (P = .002), doxycycline for 21 days (P = .02), or doxycycline for 45 days (P = .001). On day 55, C psittaci nucleic acid was detected in choanal-cloacal swabs collected from all the control birds, whereas 2 birds (28%) treated with doxycycline for 21 days and 1 bird (14%) treated with azithromycin were PCR positive. There were no significant differences between the different antibiotic treatments (azithromycin versus doxycycline) (P = .6) or the duration of the treatment (21 days versus 45 days) (P = .3). Results of PCR testing of choanal-cloacal swabs differed significantly from those of the blood samples (P = .005). On day 55, 29% of the choanal-cloacal samples (7/24) were positive, whereas only 3.7% of the blood samples (1/24) were positive.

The clinical signs observed in all the birds after inoculation involved the respiratory, gastrointestinal, nervous, and ocular systems. Deaths were recorded in 6 birds after inoculation or during the treatment period. Single deaths were recorded in groups 1 (azithromycin) and 2 (21-day doxycycline), and 2 deaths each in groups 3 (45-day doxycycline) and 4 (control). In all groups, the deaths occurred either before or within 2 days after initiation of treatment at 16 days postinoculation. The birds in the control group remained clinical throughout the study.

The plasma concentrations of doxycycline exceeded 1 [micro]g/mL at all time points tested. Mean [+ or -] standard error concentrations were 8.03 [+ or -] 1.20 [micro]g/mL and 7.41 [+ or -] 0.85 [micro]g/mL on day 14 after initiating treatment for groups 2 and 3, respectively, and 2.83 [+ or -] 0.69 [micro]g/mL and 3.81 [+ or -] 0.31 [micro]g/mL on day 21 after initiating treatment for groups 2 and 3, respectively. Plasma concentrations ranged from 0.97 to 10.89 [micro]g/mL.

At 10 days post-corticosteroid treatment, the control group and the treatment groups differed significantly in the C psittaci status determined by PCR testing of combined choanal-cloacal swab and blood samples (P = .001). Control birds were significantly more likely to be positive than the birds treated with azithromycin for 21 days (P = .002), doxycycline for 21 days (P = .001), and doxycycline for 45 days (P = .001). All the control birds were positive for C psittaci nucleic acid by PCR testing, whereas all of the treatment birds were negative.

At 30-days post steroid treatment, there also was a significant difference in the control group and the treatment groups in the C psittaci status of birds determined by PCR testing of combined choanal-cloacal swab and blood samples (P = .001). The control group was significantly more likely to be positive for C psittaci nucleic acid than the birds treated with azithromycin for 21 days (P = .005), doxycycline for 21 days (P = .003), and doxycycline for 45 days (P = .005). All of the control birds were positive, whereas all of the treatment birds were negative.

The carcasses of the 6 birds that died during the inoculation period or at the beginning (first 2 days) of the treatment period and 1 carcass of a bird from the azithromycin group (group 1) that was euthanatized at the end of the treatment period were not submitted for necropsy. Of the remaining 23 birds, spleen and liver samples collected from all the birds in the 4 groups were negative for C psittaci by PCR (2 spleens were not collected at necropsy and, therefore, not tested). At necropsy, pale livers were observed in 2 of 4 birds from group 4 (control), 1 of 6 birds from group 1 (azithromycin), and 2 of 6 birds from group 3 (45-day doxycycline). One bird in the control group had multiple pale cream-to-white nodules on the liver. In addition, 1 bird from group 2 (21-day doxycycline) had a mottled spleen, and 1 bird from group 3 (45-day doxycycline) had yellow fluid within the coelomic cavity and cream-to-white nodules on the liver, which corresponded to portal hepatitis. On histologic examination, hepatocellular vacuolar degeneration was seen in 6 of 6 birds from group 1 (azithromycin), 4 of 7 birds from group 2 (21-day doxycycline), and 5 of 6 birds from group 3 (45-day doxycycline) for 45 days, 4 of 4 birds from group 4 (control) (Table 2). Hepatic fibrosis that was graded as moderate or greater was present in 1 of 6 birds from group 1 (azithromycin), 0 of 7 birds from group 2 (21-day doxycycline), 2 of 6 birds from group 3 (45-day doxycycline), and 1 of 4 birds from group 4 (control birds). Splenic histiocytosis was present in 3 of 3 spleens examined from birds in group 1 (azithromycin) (3 marked, 1 spleen not found at necropsy, 2 spleens had insufficient tissue for both histopathology and PCR); 7 of 7 spleens from group 2 (21-day doxycycline) (5 marked, 2 moderate); 4 of 6 spleens from group 3 (45-day doxycycline) (3 marked, 1 moderate, 2 insufficient tissue for histopathology and PCR); and 3 of 4 spleens from group 4 (control) (3 marked, 1 not found at necropsy). Results of Gimenez staining of tissue samples of liver and spleen were negative in all the birds in the treatment and control groups.

Discussion

In the present study, 3 different regimens for treating C psittaci infection in cockatiels were evaluated. The morbidity, mortality, and PCR-based results confirmed that the experimental infection was successful. The results of this study indicated that C psittaci infections were effectively eliminated from all the birds in the treatment groups, regardless of the antibiotic (azithromycin or doxycycline) or, with doxycyline, the duration of treatment (21 or 45 days). Successful elimination of the pathogen is based on the failure to activate infection after immunosuppressive corticosteroid treatment, differences in gross and histologic lesions, and the absence of detectable C psittaci nucleic acid after treatment. These findings suggest that the time required to treat infected birds could be shortened significantly (>50%), which may improve compliance by pet owners and veterinarians.

In this study, clinical disease was established after inoculation with viable C psittaci organisms, and morbidity and mortality were recorded. One bird in the doxycycline group and 1 bird in the azithromycin group that were in advanced stages of disease after inoculation died either before or within 2 days after treatment began. However, in the remaining birds in the treatment groups, the clinical signs resolved, and mortality ceased after treatment was initiated with doxycycline or azithromycin. The absence of clinical signs or mortality, even after presumptive induction of an immunocompromised state with corticosteroid treatment, was consistent with elimination of the infection.

The in vitro activity of different antibiotics against C psittaci has been studied, and sensitivity to several fluoroquinolones, tetracyclines, and macrolides has been demonstrated, including both antibiotics evaluated in this study. (30,33-35) Chlamydiaceae antimicrobial resistance to tetracycline is rare, although a resistant strain of C psittaci recovered from ducks was reported. (36) There are no reports of C psittaci resistance to azithromycin in birds. The mechanisms associated with macrolide resistance in Chlamydiaceae affects the infectivity of the organisms, and, in theory, this should limit the emergence of resistant strains. (37) In vitro studies that documented the resistance of chlamydial organisms within monocytes and lymphocytes to treatment are not directly comparable with in vivo conditions. (38,39) The antibiotics that have been used successfully in vivo are tetracyclines and fluoroquinolones. (40,41) Although results of early experimental studies suggested enrofloxacin as a potential alternative treatment, (40,41) anecdotal use of enrofloxacin in clinical practice showed that many birds fail to eliminate infection. (12) Besides their antimicrobial activity, macrolides and tetracyclines possess independent anti-inflammatory properties; however, more research is needed to determine whether this occurs in avian species. (42,43)

For more than a decade, doxycycline has been the antibiotic of choice for treating avian chlamydiosis. Doxycycline alters the replication of the reticulate body by inhibiting the synthesis of enzymes, the growth and fission of the reticulate bodies, and possibly the reorganization of the elementary bodies. (3) Antimicrobial-induced damage that occurs may be temporary, with the organism resuming normal replication within 5.5 days after stopping therapy? In vitro, this drug is not effective in treating persistently infected cells in which the organism is not replicating. (3) Plasma concentrations >1 [micro]g/mL for the duration of the treatment period are thought to inhibit C psittaci replication. (12,15-20,44) Plasma concentrations of doxycycline in the birds in our study ranged from 0.97 to 10.89 [micro]g/mL. Although the plasma concentrations that were measured were much higher than those recommended for the treatment of avian chlamydiosis, no toxic effects were observed. An even lower dosage (eg, 25 mg/kg q24h) may establish adequate plasma concentrations, but this requires additional study to confirm. Doxycycline treatment periods of 30 to 45 days are thought necessary to eliminate C psittaci infection, (13,14,44) but maintenance of blood concentrations as low as 0.1 [micro]g/mL and treatment periods as short as 3 weeks were effective in eliminating infection from experimentally infected Amazon parrots (Amazona viridigenalis). (45)

Macrolide antibiotics, such as azithromycin and clarithromycin, have been used for years to treat humans with Chlamydia trachomatis or Chlamydophila pneumoniae infections. (9,46) These drugs are well tolerated and have a prolonged tissue half-life. Studies in humans demonstrated that a single dose of azithromycin is as effective in eliminating C trachomatis infection as a 7-day course of doxycycline. (47,48) Similar studies demonstrated that a 10-day treatment with erythromycin or clarithromycin, and a 5-day course of azithromycin are effective for the treatment of C pneumoniae respiratory infections, but prolonged therapy (eg, at least 2 to 3 weeks) are desirable, because recrudescent symptoms have been reported. (9)

The PCR techniques available in the diagnosis of C psittaci infections are based on detection of DNA sequences of the MOMP gene, pmp genes, or the 16S-23S ribosomal RNA. (49-53) Various PCR-based assays have been used to detect C psittaci nucleic acid in swabs (eg, conjunctival, choanal, pharyngeal, and cloacal), feces, tissue biopsy, and whole-blood samples, and postmortem in tissues, cell cultures, and embryonated eggs. (53-55) A PCR-based test was selected for this project because it is considered the most sensitive and specific test available and can provide rapid results? (56-58) Chlamydophila psittaci nucleic acid was detected in swab samples from 1 cockatiel in group 1 (azithromycin) and 2 cockatiels in group 2 (21-day doxycycline) on day 55 after starting antibiotic treatment. The PCR-based assays do not differentiate between viable and nonviable nucleic acid, and because none of these 3 birds were positive 30 days after administration of dexamethasone or at necropsy, the nucleic acid may have been present because of residual nucleic acid in the cells. Contamination of the choanal-cloacal swabs from environmentally derived sources of target nucleic acid is also possible. Choanal-cloacal swabs should always be considered an environmental swab, and positive results do not differentiate between indogenous and exogenous sources of target nucleic acid. Assays to detect messenger RNA would be required to differentiate between viable and nonviable nucleic acid in clinical samples.

To evaluate the possibility of posttreatment persistence of C psittaci, corticosteroids were used to induce presumed immunosuppression. Extreme environmental changes or concurrent infections may activate persistent infections through stress and subsequent immunosuppression, which result in clinical disease. (3,8) However, birds that recover from C psittaci infections are susceptible to reinfection, and results of previous studies that suggest the possibility of a persistent infection failed to control for reinfection rather than reactivation of a quiescent infection. Quantifying the number of organisms shed by these birds before and after treatment may have provided additional information. Neither the whole blood nor the choanal-cloacal swabs from the birds treated with antibiotics were positive for C psittaci nucleic acid after corticosteroid treatment. This finding suggests that these animals had stopped shedding detectable levels of C psittaci nucleic acid and that presumed immunosuppression did not reactivate an infection or shedding of nucleic acid to detectable levels.

Chlamydophila psittaci DNA was not recovered from the spleen or liver samples from any of the birds at the end of the study. These results could be associated with the sensitivity of the assay or the absence of the organism in those tissues. The PCR assay used for the liver and spleen samples at necropsy was different from the assay used for the choanal-cloacal and blood samples. Possibly, the necropsy specimens were negative for C psittaci nucleic acid because the organism was located elsewhere. Unfortunately, tissues other than liver and spleen were not evaluated at the time of the necropsy. Future studies should consider sampling a wider range of tissues.

The histopathologic lesions described in the birds from this study are consistent with those described for birds infected with C psittaci?59,60 The microscopic findings are mostly nonspecific, except for the presence of Levinthal-Cole-Lillie bodies, which are pathognomonic. Levinthal-Cole-Lillie bodies can occur in many organs but are especially common in serosal membranes? The single most consistent histopathologic feature of lesions associated with C psittaci infections in birds is hystiocytosis, an increased number and size of cells in the hystiocyte-macrophage system, either in the interstices of parenchymatous organs or as the predominant cellular component of fibrinous exudates on air sac and serosal surfaces. (5) Typical of more acute disease is the intrasinusoidal proliferation of Kupffer star cells (pearl string-like appearance) in the liver. In the liver, intrasinusoidal histiocyte (Kupffer cell) hyperplasia, often accompanied by local or diffuse mononuclear cell infiltration, is the common manifestation of subacute-to-chronic chlamydiosis of mild-to-moderate severity. Slight-to-marked intrahepatocellular bile stasis and accumulation of iron pigment are frequent sequelae of hepatitis. Multiple foci of coagulation necrosis of histiocytes parenchyma has been described as a prominent lesion that results from experimentally induced avian chlamydiosis in parrots and may be the only lesion observed grossly or histologically in the acute fatal diseases. Moderate-to-marked bile-duct hyperplasia in recurrent, subacute episodes of avian chlamydiosis may be present over a period of many months or even years. (61) In the spleen, hyperplasia of histiocytes in the perivascular sheaths of the arterioles, with consequent thickening of the sheaths, is common in parrots with clinical signs of chlamydiosis. The degree of splenomegaly evident is generally quantitatively proportional to the number of histiocytes present. Depletion of lymphocytes from the white pulp is common; it may be a relative decrease, but, in many cases, the lymphocyte paucity is absolute and accompanied by absolute plasmacytosis. (61)

In this study, few gross or histologic differences were observed between untreated infected birds and infected birds treated with doxycycline or azithromycin. On histologic examination, hepatic vacuolar degeneration and splenic histiocytosis were present within all groups, regardless of treatment. Hepatocellular vacuolar degeneration is a common, nonspecific change in the liver that may or may not be related to C psittaci infection. Splenic histiocytosis, as noted above, has been associated with C psittaci infection, however, it is not known how long the histiocytosis persists after infection has been cleared. Because all birds in the study were infected initially, the splenic histiocytosis in the spleens of doxycycline- and azithromycin-treated birds may have been residual.

Results of Gimenez staining of hepatic and splenic tissues were negative in all treatment and control groups. The control for the stain, a C psittaci positive bird not included in the study, stained positive. These findings suggest the absence of C psittaci in these tissues. Direct visualization of chlamydial organisms in smears and paraffin-embedded tissue sections can be performed by several techniques. Histochemical staining with Gimenez, (62) modified Gimenez, (63) Macchiavello,64 Stamp, (65) and Giemsa66 are techniques used to demonstrate the characteristic chlamydial intracytoplasmic inclusions present in impression smears made from swabs, removed tissues, or yolk-sac membranes after isolation. Sensitivity and specificity of Gimenez staining was found to be 100% and 85%, respectively, when compared with culture of visceral organ tissue from dead birds. (67) Immunohistochemical staining is another method for detecting chlamydiae in cytologic and histologic preparations, and is more sensitive and specific than histochemical techniques. (68)

When selecting a chlamydial treatment regimen for an avian patient, several factors must be considered in addition to the duration of treatment and drug type, such as adverse effects, cost, and governmental regulations. There were no adverse effects reported in any of the birds in the treatment groups in this study. The most common adverse effect associated with oral doxycycline therapy in birds is regurgitation and vomiting. (12) Currently, no adverse effects have been reported with oral azithromycin in birds. The cost difference may be an important factor when treating a large number of birds, and azithromycin is a more expensive drug. The governmental regulations in some countries may require longer treatment than the 21 days suggested from the results of this study. The Compendium of Measures to Control Chlamydophila psittaci Injection Among Humans (Psittacosis) and Pet Birds" (Avian Chlamydiosis) 10 should be followed in the United States, and governmental regulations might guide treatment in other countries.

Although the results of this experiment are encouraging, the limitations of this study should be considered before extrapolating these results to field use. First, the standards for treatment success are higher for C psittaci than for some other avian diseases. It is important to eradicate the infection to prevent reinfection in exposed birds. Subtherapeutic treatment could also lead to human exposure and the generation of resistant strains of C psittaci. Second, an acute infection in a limited number of birds was treated in this study. As noted in the introduction and discussion, chronic or persistent chlamydial infections may be more difficult to eradicate and may require longer treatment times in some individual birds. Also, a single strain of chlamydiae was used for infection, and it is possible that other strains could be more difficult to treat. Finally, these birds were housed under optimal environmental conditions. Birds housed under poor conditions or fed deficient diets may show different results. Further testing of infections under field conditions should be done with appropriate testing to make sure the infection has cleared before exposing treated birds to other birds and humans.

The results of this study suggest that C psittaci can be eliminated from experimentally infected cockatiels by using either doxycycline or azithromycin at shorter treatment regimens than has been historically used. A shorter treatment period may increase compliance, reduce the stress of treatment on the birds and their care providers, reduce the possibility for drug reactions, decrease the amount of environmentally discharged antimicrobials, and reduce the cost of treatment. Additional studies are needed to determine the effectiveness of these results in naturally infected birds.

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David Sanchez-Migallon Guzman, LV, MS, Dipl ECZM (Avian), Orlando Diaz-Figueroa, DVM, MS, Dipl ABVP (Avian), Thomas Tully Jr, DVM, MS, Dipl ABVP (Avian), Dipl ECZM (Avian), Paula Ciembor, DVM, PhD, Tim Morgan, DVM, Phi), Dipl ACVP, Michael Walden, MS, DVM, Phi), Robert P. Poston, MS, Keven Flammer, DVM, Dipl ABVP (Avian), Mark A. Mitchell, DVM, MS, Phi:), and Branson Ritchie, DVM, Phi:), Dipl ABVP (Avian), Dipl ECZM (Avian)

From the Department of Veterinary Clinical Sciences (Guzman, Diaz-Figueroa, Tully, Mitchell), Department of Pathobiological Sciences (Morgan, Walden), Louisiana Veterinary Medical Diagnostics Laboratory (Poston), Louisiana State University, School of Veterinary Medicine, Baton Rouge, LA 70803, USA; Emerging Diseases Research Group, University of Georgia, College of Veterinary Medicine, Athens, GA 30602, USA (Ciembor, Ritchie): and the Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough St, Raleigh, NC 27606, USA (Flammer). Present address: Department of Surgical Sciences, University of Wisconsin, School of Veterinary Medicine, 2015 Linden Dr, Madison, WI 53706 1100, USA (Guzman); Lake Howell Animal Clinic, 856 Lake Howell Rd, Maitland, FL 32751, USA (Diaz- Figueroa); Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, 1008 W Hazelwood Dr, Urbana, IL 61802, USA (Mitchell).
Table 1. Results of PCR testing for C psittaci nucleic acid (reported
as positive/negative) from combined choanal cloacal swab, blood,
liver, and spleen samples from cockatiels inoculated with C psittaci.
There were 3 different treatment groups and 1 control group.

                                   Treatment day (a)

                         0    55    85    105       105     105

Treatment (group no.)   Choanal-cloacal + blood    Liver   Spleen

Azithromycin, 21 d (1)  8/0   1/6   0/7   0/7       0/6     0/6
Doxycycline, 21 d (2)   8/0   2/5   0/7   0/7       0/7     0/7
Doxycylcine, 45 d (3)   8/0   0/6   0/6   0/6       0/6     0/6
Positive control (4)    6/0   4/0   4/0   4/0       0/4     0/4

(a) Day 0 (16 days postinoculation, antibiotic treatment starts);
day 55 (10 days after finishing 45 days treatment); day 85 (10 days
post-dexamethasone treatment administration); day 105 (30 days
post-dexamethasone treatment administration, euthanasia, and
postmortem examination).

Table 2. Histopathologic findings in livers and spleens of cockatiels
inoculated with C psittaci and treated with azithromycin at 40 mg/kg
PO g48h (group 1) for 21 days, doxycycline at 35 mg/kg PO g24h for
21 days (group 2), doxycycline at 35 mg/kg PO g24h for 45 days
(group 3), and positive controls (group 4) during treatment for the
different groups of birds.

                                   Liver                Spleen

                                       Moderate    Moderate    Marked
                            Vacuolar   or marked   histiocy-   histio-
Treatment                    change    fibrosis      tosis     cytosis

Azithromycin, 21 d (n = 6)     6           1           3          0
Doxycycline, 21 d (n = 7)      4           0           5          2
Doxycycline, 45 d (n = 6)      5           2           3          1
Positive control (n = 4)       4           1           3          0
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Title Annotation:Original Studies
Author:Guzman, David Sanchez-Migallon; Diaz-Figueroa, Orlando; Tully, Thomas, Jr.; Ciembor, Paula; Morgan,
Publication:Journal of Avian Medicine and Surgery
Article Type:Report
Geographic Code:1USA
Date:Mar 1, 2010
Words:7607
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