Materials and Methods

Birds—Eighteen cockatiels that were between 6 and 18 months old and of either sex and that had been purchased from an aviculturist were used in the study. The birds were moved to the North Carolina State University, College of Veterinary Medicine, Laboratory Animal Research facility and allowed to acclimate for 45 days before the study was initiated. During this acclimation period, the birds were examined, and blood samples were collected to determine PCV and total solids concentration. In addition, swab specimens from the cloaca and choana were submitted for aerobic bacterial and fungal culture. Six randomly selected birds were tested for Chlamydophila psittaci DNA. In brief, a swab was touched to the conjunctiva, choana, and cloaca of each bird and tested for C psittaci DNA by a commercial laboratory.^a

Birds were fed a diet containing equal volumes of pellets^b and a seed mix.^c Spray millet was offered as a treat on days when the birds were captured or moved. Room temperature was maintained between 20° and 24°C (68° and 76°F), and lighting was controlled in a 12-hour cycle. The experimental protocol was approved by the institutional animal care and use committee.

Study design—The study was designed as a randomized controlled clinical trial. Eleven of the cockatiels were randomly assigned to a treatment group, and the remaining 7 were assigned to the control group; assignments were made on the basis of the order birds were captured and removed from the transport carrier. To avoid cross contamination, birds in the control group were moved to a room in a separate building and cared for by personnel different from those who cared for birds in the treatment group. In addition, on days that samples were collected, birds in the treatment group were handled first, and individuals collecting samples washed their hands with antibacterial soap and donned new shoe covers and laboratory coats before entering each bird room.

For birds in the treatment group, the drinking water was medicated with doxycycline hyclate for 30 days. Four 100-mg doxycycline hyclate capsules^d were added to 1,000 mL of tap water to create a solution assumed to contain 400 mg of doxycycline/L. The solution was mixed with a magnetic stirrer for 4 minutes and stored in a polypropylene bottle at 20°C until used; fresh solution was made every 24 to 36 hours. Prior to dispensing, the bottle was shaken for at least 30 seconds. Water was offered ad libitum in open stainless steel bowls. Birds in the control group received unmedicated tap water.

Birds in both groups were observed daily, and activity level and characteristics of the droppings were rated as normal or abnormal. In addition, birds in both groups were examined and weighed 40, 35, and 19 days before the initiation of treatment; on days 0, 7, 14, 21, and 28 during treatment; and 4, 25, and 28 days after treatment was discontinued. During these examinations, condition of the oropharynx was scored on a scale from 0 to 4 (0 = normal; 1 = slightly red; 2 = moderately red; 3 = markedly red; and 4 = markedly red with erosions or excess mucus production), and condition of the nares was scored on a scale from 0 to 3 (0 = normal; 1 = solid material in nares; 2 = solid material in nares with mild swelling or redness; and 3 = solid material in nares with enlargement of the opening). All physical examinations were performed by 2 individuals (EEE and KF).

Swab specimens of the choana and oropharynx were collected from all birds for detection of spiral bacteria 40, 35, and 19 days before the initiation of treatment; on days 0, 7, 14, 21, and 28 during treatment; and 4 and 28 days after treatment was discontinued (data for swab specimens collected on day 0 were lost). Additional swab specimens were obtained from birds in the treatment group on day 3 of treatment and 160 days after treatment was discontinued. For collection of swab specimens, a small swab^e was inserted deep inside the choanal slit and then wiped across the mucosal surface of the oropharynx.

Packed cell volume; plasma total solids, albumin, total bile acids, calcium, glucose, phosphorus, total protein, and uric acid concentrations; and plasma aspartate aminotransferase and creatine kinase activities were assessed in 7 birds in the treatment group on days 0, 7, 14, and 21. Biochemical analyses were performed with an automated analyzer^f; birds with abnormal test results were retested 4 days after treatment ended. Blood samples for determination of plasma doxycycline concentrations were collected between 9:00 and 10:45 AM from 7 randomly selected birds in the treatment group on days 7, 21, and 28 and from 4 birds on day 14.

Following the end of the treatment period, birds in the control group were held for an additional 13 days. To evaluate treatment under field conditions, 6 birds from the control group were returned to the breeder (the remaining bird was adopted as a pet). After a reacclimation period of 45 days, the breeder was instructed to treat the birds with water medicated with doxycycline for 21 days. The owner prepared fresh solution daily by adding the contents of four 100-mg doxycycline hyclate capsules to 1 L of water in a bottle and shaking for at least 60 seconds. Birds were tested for spiral bacteria on the day treatment started, the day treatment ended, and 30 days after treatment ended. Physical examinations, but no other testing, were also performed on these dates.

Examination of swab specimens for spiral bacteria—Material from each choanal and oropharyngeal swab specimen was transferred to 2 microscope slides by rolling half of the swab on 1 slide, and half on the other. This process was repeated, so that each side of the swab was rolled once on each slide. One slide was then stained with Gram stain, and the other slide was stained with a Romanowsky stain.^g

Slides for Gram staining were heat-fixed and stained by means of standard methods, except that only brief contact with the decolorization solution was used to avoid overdecolorizing. Slides for Romanowsky staining were dipped in methanol fixative 2 times, dipped in methylene blue stain 8 times, dipped in eosin stain 10 times, and dipped in deionized water 5 times.

Slides were examined at 100X and 1,000X, and the number of spiral bacteria was counted in ten 100X fields. The count was then converted to a score from 0 to 3 (0 = no spiral bacteria seen; 1 = 10 to 19 spiral organisms/10 fields; 2 = 20 to 30 organisms/10 fields; and 3 = > 30 organisms/10 fields). Birds were considered positive for spiral bacterial infection if spiral bacteria were detected (ie, score of 1, 2, or 3) by either staining method.

All slides were examined by a single individual (EEE). To assess the accuracy of infection classifications, stained smears that had been obtained from all birds on treatment day 21 and that had been obtained 160 days after treatment from birds that had been treated with doxycycline were sent to another individual (LLW) who scored all slides as positive or negative for the presence of spiral bacteria. This individual was blinded to the identification of the smears and to the results of the first evaluator.

Measurement of plasma doxycycline concentration—Plasma doxycycline concentrations were determined by means of high-pressure liquid chromatography with UV detection. A validated protein precipitation method⁶ was used to prepare plasma samples for analysis. Pooled plasma samples from cockatiels that had not been treated with doxycycline were used as a control and to check for interfering peaks. The limit of quantification was 0.25 μg/mL.

Statistical analysis—Paired t tests were used to compare lesion scores among days. All analyses were performed with standard software.^h Values of P < 0.05 were considered significant.

Results

Baseline evaluations—In all 18 birds, spiral bacteria were seen in swab specimens from the choana and oropharynx obtained prior to the initiation of treatment (Figure 1). Condition of the nares was scored as 1 (solid material in nares) in 5 of the 18 birds, as 2 (solid material in nares with mild swelling or redness) in 10, and as 3 (solid material in nares with enlargement of the opening) in 3. Condition of the oropharynx was scored as 0 (normal) in 3 of the 18 birds, as 1 (slightly red) in 13, and as 2 (moderately red) in 2. None of the birds had generalized signs of illness (eg, lethargy or inappetence). Aerobic bacterial and fungal culture of swab specimens from the cloaca and choana did not yield any gram-negative bacteria or yeast, and PCV and total solids concentration prior to the initiation of treatment were within reference limits. Results of tests for C psittaci DNA were negative.

Concordance of cytologic examination results— Results of cytologic examination of choanal and oropharyngeal swab specimens (n = 111) collected from all birds 35 and 19 days before the initiation of treatment, from all birds on days 7 and 14 of treatment, from the treatment-group birds on day 3 of treatment, and from the control-group birds on days 21 and 28 of treatment and 4 and 28 days after treatment ended were examined to determine concordance between results of examination of smears stained with Gram stain and results of examination of smears stained with a Romanowsky stain. For 95 of the 111 (86.6%) paired specimens, results were concordant. For 10 (9.0%) specimens, spiral bacteria were seen during examination of the smear stained with Gram stain, but not during examination of the smear stained with a Romanowsky stain. For 6 (5.4%) specimens, spiral bacteria were seen during examination of the smear stained with a Romanowsky stain but not during examination of the smear stained with Gram stain.

When stained smears that had been obtained from all birds on treatment day 21 and from treatment-group birds 160 days after treatment ended were sent to a second individual (LLW) who scored all slides as positive or negative for the presence of spiral bacteria, there was 100% agreement between results of this evaluator and results of the initial evaluator (EEE).

Clinical trial—Mean ± SD plasma doxycycline concentrations were 2.86 ± 1.34 μg/mL, 2.70 ± 0.83 μg/mL, 2.55 ± 0.82 μg/mL, and 2.26 ± 1.0 μg/mL on treatment days 7, 14, 21, and 28 respectively. Overall, plasma doxycycline concentration ranged from 0.83 to 4.39 μg/mL.

Spiral bacteria were identified in swab specimens from all 11 birds in the treatment group 40, 35, and 19 days prior to initiation of treatment and on treatment days 3 and 7. In addition, spiral bacteria were seen in 10 of the 11 birds on treatment day 14. However, spiral bacteria were not seen in specimens from any of the treatment group birds on treatment days 21 and 28 or 4, 25, 28, and 160 days after treatment was discontinued. All 7 birds in the control group were positive for spiral bacteria on all observation days (ie, 40, 35, and 19 days prior to the initiation of treatment; days 7, 14, 21, and 28 of treatment; and 4, 25, and 28 days after treatment was discontinued).

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Photomicrographs of Gram-stained smears collected from the choanal slit of cockatiels, showing higher (A) and lower (B) numbers of spiral bacteria. Solid bar = 10 μm.

Citation: Journal of the American Veterinary Medical Association 232, 3; 10.2460/javma.232.3.389

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For the 11 birds in the treatment group on day 0, condition of the oropharynx was scored as 0 in 3 birds, as 1 in 7 birds, and as 2 in 1 bird, and condition of the nares was scored as 1 in 3 birds, 2 in 7 birds, and 3 in 1 bird. Twenty-eight days after treatment ended, score for condition of the oropharynx was improved in 9 of the treatment-group birds, unchanged in 1, and worse in 1, compared with scores on day 0. Score for condition of the nares was improved in 7 birds, unchanged in 2 birds, and worse in 2 birds. When day 28 scores were compared with day 0 scores, scores for condition of the oropharynx were significantly (P = 0.035) improved, but scores for condition of the nares were not (P = 0.32). For birds in the control group, there were no significant changes in scores for condition of the oropharynx or condition of the nares after treatment, compared with scores prior to treatment.

No clinically important adverse effects associated with treatment were identified. Throughout the treatment period, activity level, behavior, and droppings of the birds were normal. Seven of the 11 treatment-group birds maintained or gained weight, and 4 lost weight; mean ± SD percentage change in weight was −2.2 ± 6.7% (range, −13.4% to 6.9%). One of the 7 controlgroup birds gained weight, and 6 lost weight; mean percentage change in weight was −9.4 ± 7.3% (range, −16.6% to 1.5%). Packed cell volume, plasma total solids concentration, and results of plasma biochemical analyses were within reference limits in the treatmentgroup birds before treatment, except that 1 bird had a slightly high aspartate aminotransferase activity (551 U/L; reference range, 107 to 481 U/L) and PCV (61%; reference range, 43% to 57%). The only clinically important abnormalities identified after day 0 were slightly high aspartate aminotransferase activities (485 and 496 U/L) on day 21 in 2 birds. All values were within reference limits when these birds were retested 4 days after treatment ended.

The 6 control birds that were returned to the breeder all tested positive for spiral bacteria prior to treatment with doxycycline and negative for spiral bacteria on day 21 of treatment and 30 days after treatment ended. The breeder did not observe any abnormalities, and results of physical examinations conducted by one of the authors (EEE) were unremarkable.

Discussion

Results of the present study suggested that providing drinking water to which doxycycline had been added at a concentration of 400 mg/L was effective in eliminating spiral bacterial infections in cockatiels. The medicated water was well-accepted, maintained fairly consistent plasma doxycycline concentrations in birds, and was associated with few adverse effects. Drug delivery was easier than oral administration, which would have required capturing the birds twice daily.

Daily mean plasma doxycycline concentrations in treated birds in the present study ranged from 2.55 to 2.86 μg/mL. The minimum concentration of doxycycline that will inhibit growth of spiral bacteria in cockatiels is not known because the organism has not yet been successfully cultured. For comparison, however, in vitro minimum inhibitory concentrations of doxycycline are 0.12 to 2.0 μg/mL for Helicobacter pullorum⁷ and 0.023 to 0.125 μg/mL for Helicobacter pylori.⁸ In people, H pylori is often treated with combinations of 2 or 3 antimicrobials to improve efficacy and avoid induction of resistance. Treatment with doxycycline alone was apparently successful in the birds treated in the present study; however, further study is needed to determine if monodrug therapy is consistently effective in cockatiels.

Treatment was associated with minimal adverse effects in the present study, and birds had normal activity levels and behavior throughout the treatment period. Weight loss was greater in the control group than the treatment group probably because the control group was moved to another building at the start of the trial. It is also possible that prolonged spiral bacterial infection adversely affected body weight. The transient, mild increase in aspartate aminotransferase activity in 3 birds was considered clinically unimportant.

For 16 of 111 (13.4%) specimens in the present study, results of examination of smears stained with Gram stain did not agree with results of examination of smears stained with a Romanowsky stain. Because for each specimen a single swab was used to inoculate the 2 slides, this disagreement may have been due to differences in the number of organisms deposited on the 2 slides during cytologic preparation. A standard method for identifying spiral bacterial infection has not been developed. The sensitivity of cytologic examination of smears stained with Gram stain or a Romanowsky stain is unknown, and it is possible that treated birds in the present study remained infected with spiral bacteria that might have been detected with other methods. Improved methods of antemortem diagnosis should be investigated, including culture of the organism, cytologic examination of silver-stained smears, and development of a specific PCR test.

Clinical signs of infection at the time birds in the present study were acquired were generally mild, with 3 birds having moderate signs of infection. Although the same investigators examined and scored lesions in all birds before, during, and after treatment, it was difficult to consistently scores lesions because there was normal variation in oral cavity and nasal color between birds (especially between birds with different color mutations), individuals varied on different days, and changes were mild. Changes could have been more reliably documented if the birds had been sequentially photographed under standardized lighting conditions, but this was not done. Therefore, our assessment of clinical signs before and 28 days after treatment should be considered subjective and restricted to the observation that oral cavity redness resolved in most birds with treatment but the mild redness and enlargement of the nares did not in 4 birds. It is possible that packing of feather debris in the nares of 2 of these birds accounted for the nasal irritation. The clinical importance of mild redness of the oral cavity is debatable and may be caused by factors other than disease (eg, excitement and repeated vocalization).

The organism causing spiral bacterial infections in cockatiels has not been characterized, and it is uncertain whether all spiral bacterial infections in cockatiels are caused by the same organism. A preliminary report suggests that at least 1 spiral bacteria isolate found in a cockatiel may be a member of the genus Helicobacter.³ To our knowledge, there is only a single published report of Helicobacter infection in psittacine birds other than cockatiels. In that report,⁹ H pullorum infection in a blue bonnet parrot (Psephotus haematogaster) was associated with bloody diarrhea and responded to treatment with doxycycline.

Providing drinking water medicated with doxycycline has also been suggested as a method for treating C psittaci infections in cockatiels, and a previous study¹⁰ found that providing drinking water to which doxycycline had been added at concentrations of 280 and 830 mg/L was associated with mean ± SD plasma doxycycline concentrations of 1.45 ± 0.37 μg/mL and 5.37 ± 2.49 μg/mL, respectively. Maintenance of plasma concentrations near 1 μg/mL has been found to be efficacious for treatment of C psittaci, so it is likely that both spiral bacterial infection and psittacosis could be treated simultaneously with medicated drinking water. Medicated drinking water would be advantageous for treatment of cockatiels in aviculture collections and could be offered for sale at pet stores. Plasma concentrations achieved with drinking water medicated with doxycycline vary among psittacine species,^11,12,i so the dose used in cockatiels may not be effective in other birds.

Although treatment was apparently successful in these cockatiels, care should be taken in extrapolating this treatment method to other groups of cockatiels. Immunocompetence, concurrent disease, diet, environmental conditions, and supportive care could influence treatment success in individual birds, so additional studies are needed in other groups of cockatiels. Plasma drug concentrations may also vary with water consumption, and breeding birds and birds housed outdoors in hot weather may consume greater amounts of water than the birds in the present study. Doxycycline is less affected by cation chelation than other tetracyclines; however, high dietary calcium contents may reduce absorption,ⁱ and birds provided with a mineral block or cuttle bone may have lower plasma concentrations.