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Assessment of plasma concentrations and potential adverse effects of doxycycline in cockatiels (Nymphicus hollandicus) fed a medicated pelleted diet.

Abstract: Doxycycline hyclate was mixed with soybean oil and then added to a low-fat pelleted diet that contained approximately 2.4% fat, which produced a final diet that contained a calculated 6.4% fat and 300 mg doxycycline per kilogram of diet. The medicated diet was fed to 9 healthy adult cockatiels (Nymphicus hollandicus) for 47 days; a control group of 6 birds received the identical diet without doxycycline. Trough doxycycline plasma concentrations were measured 7 times during treatment and ranged from 0.98 to 3.83 [micro]g/mL with an overall median of 2.09 [micro]g/mL. The birds were observed daily, weighed, and examined at least weekly, and selected plasma biochemical parameters were measured before treatment and at days 21 and 42. No adverse effects were noted, except one treatment bird became obese. This medicated diet may be suitable for treating spiral bacteria and Chlamydophila psittaci infections in cockatiels that will consume a pelleted diet.

Key words: Chlamydophila psittaci, spiral bacteria, antimicrobial drug, doxycycline, pharmacology, avian, cockatiel, Nymphicus hollandicus


Doxycycline is a semisynthetic tetracycline antibiotic that shows greater bioavailability and a longer elimination half-life than older drugs, such as oxytetracycline and chlortetracycline, (1,2) which allows less drug to be used in medicated food or water, thereby improving palatability. (3-5) In companion avian medicine, doxycycline is most commonly used to treat chlamydiosis and spiral bacterial infections. (3-7)

Cockatiels (Nymphicus hollandicus) are popular companion birds and have been implicated in avian chlamydiosis outbreaks, which affect both birds and people. (3, 7-13) Avian chlamydiosis is an infectious disease of birds caused by the obligate intracellular bacterium Chlamydophila psittaci. Clinical signs of infection in birds range from inapparent to severe systemic disease and death. (3,9-12) Chlamydophila psittaci can infect people and cause flu-like symptoms, pneumonia, and, rarely, death. (8, 11-13) Therefore, control of this disease is important for both avian and human health.

Doxycycline has been used for decades to successfully treat avian chlamydiosis. Historically, public health agencies in the United States and Germany chose maintenance of blood or plasma concentrations of chlortetracycline [greater than or equal to] 1 [micro]g/mL as the pharmacologic standard for developing treatment regimens for avian species. (14-16) This standard was based on treatment studies in quarantined imported psittacine birds in which successful treatment was correlated with maintaining systemic chlortetracycline concentrations higher than 1 [micro]g/mL. (15-21) Doxycycline is presumed to be as active or more active than chlortetracycline based on its pharmacologic properties and efficacy against other bacterial species. (12) For the purpose of this study, we chose 1 [micro]g/mL as the desired minimal doxycycline concentration during treatment. Past studies have shown that this level is easily achieved in several psittacine bird species and is well tolerated. (22-27) Lower concentrations may be effective, but the risk of treatment failure and generation of microbial resistance may be greater.

Doxycycline is bacteriostatic and is presumably effective only when chlamydiae are actively replicating. (9,21) Because chlamydiae can remain dormant in host cells, prolonged treatment periods have been recommended. (9,21) In the United States, it is empirically recommended that psittacine birds other than budgerigars (Melopsittacus undulatus) be treated for 45 days, (11) and we chose this period for the purpose of our study. A recent study showed that oral administration of doxycycline for 21 days was successful in treating chlamydiosis in experimentally infected cockatiels with an acute infection, so a shorter duration of treatment may be effective for some birds. (28)

The systemic concentrations of doxycycline in cockatiels achieved when using a drug delivered per os, (22,28) by intramuscular injection, (23) and by feeding medicated seed, mash, or water (23) have been reported. Each method has advantages and disadvantages. Drug delivered per os or by injection is useful to rapidly establish therapeutic drug concentrations in birds that are reluctant to eat or drink but require capture and handling of the bird for drug administration. Use of medicated water or feed precludes the need for capture, but the bird must consume adequate food or water to maintain therapeutic drug levels. The medicated food or water must also be properly prepared, which results in potential compliance issues. Psittacine birds are often reluctant to accept novel foods, so offering a diet that is familiar to the bird is advantageous, especially because food consumption may be reduced when the birds are stressed or ill. Availability of a doxycycline-medicated pelleted diet would aid treatment of birds that consume a pelleted diet. The purpose of this study was to assess if feeding a pelleted diet medicated with 300 mg doxycycline hyclate per kilogram of feed would safely maintain trough plasma doxycycline concentrations higher than 1 [micro]g/mL.

Materials and Methods

Birds and housing

Twenty cockatiels, weighing 80-109 g, were borrowed from a local aviculturist. As described below, the experimental plan was designed for 15 birds; 5 additional birds were initially acquired to provide extras. All the birds were at least 9 months old, had been hand-raised, and had been fed a variety of diet types (eg, seed, pellets, and chopped vegetables) at the time they were weaned. After weaning, they were fed a seed-based diet supplemented with chopped vegetables. Two weeks before acquisition for this project, the aviculturist offered the birds a pelleted diet (Daily Maintenance mini pellet, Roudybush, Woodland, CA, USA) in addition to their free-choice seed and chopped vegetable diet. On the day of acquisition, the birds were examined, weighed, and separated into treatment and control groups of 10 birds each based on the order of capture from their transport boxes. In 5 birds from each group, a swab was touched to the conjunctiva, choana, and cloaca of each bird and tested for C psittaci DNA by a commercial laboratory (Chlamydophila psittaci polymerase chain reaction test, Infectious Disease Laboratory, College of Veterinary Medicine, University of Georgia, Athens, GA, USA).

The birds in the 2 groups were judged healthy on the basis of physical examination, body weight, and daily observation. Each group was housed similarly in separate rooms in the Laboratory Animal Research Facility at the College of Veterinary Medicine in 2.7 x 0.9 x 2.1-m wire flight cages and fed a free-choice diet that consisted of seed mix (Fiesta, Kaytee Products, Chilton, WI, USA) and pellets that contained approximately 8.2% fat (Daily Maintenance mini pellet; Roudybush). No cuttlebone, mineral blocks, or other nutritional supplements were offered. Room temperature was maintained between 20[degrees]C and 24[degrees]C (68[degrees]F and 76[degrees]F), and lighting was controlled in a 10-hour light and 14-hour dark cycle. The experimental protocol was approved by the Institutional Animal Care and Use Committee.

The amount of seed fed to the birds was gradually reduced over a 10-day period until the birds were offered pellets only and maintained consistent body weight. During this period, the birds and their droppings were observed daily, and body weight and physical examination parameters were assessed 3 times. On the day before starting the treatment period, the treatment group was reduced to 9 birds and the control group to 6.

A total of 90.7 kg (200 lb) of the treatment diet was prepared by suspending doxycycline hyclate powder in soybean oil and then mixing the medicated oil with a low-fat pelleted diet that contained approximately 2.4% fat (Low Fat Maintenance mini pellet; Roudybush). The process was calculated to produce a diet that contained 300 mg doxycycline per kilogram of diet and 6.4% fat. To accomplish this, the doxycycline-medicated oil was prepared in 2 equal batches to accommodate accurate measurement with available laboratory glassware. Doxycycline hyclate was collected from 165 opened 100-mg capsules (doxycycline hyclate capsules USP 100 mg, Watson Laboratories Inc, Corona, CA, USA) that weighed 69.2 g. The doxycycline powder was slowly added to 2200 g soybean cooking oil in a 5.7 L (6 qt) porcelain-lined bowl while mixing with a stick mixer (Minipimer, Braun, Brooklyn, NY, USA). To crudely assess uniformity, a portion of the suspension was placed in a 100-mL graduated cylinder and transilluminated with a 60-W light source. The suspension remained visually uniform for at least 5 minutes, after which the doxycycline powder slowly settled. Therefore, to assure uniform mixing, the doxycycline-oil suspension was remixed for at least 30 seconds before transferring any amount. A total of 11.3 kg (25 lb) of pellets was placed in the drum of a small cement-mixing machine (Portable Cement Mixer with poly drum, 2 cubic feet, model PCM185, Northern Tool and Equipment, Burnsville, MN, USA). While the drum was turning, 473 g of the doxycycline-oil mixture was slowly poured over the pellets and mixed for 6 minutes. The doxycycline-oil mixture was absorbed by the pellets, and there was no excess oily residue. Each of the four 11.3-kg batches was equally distributed by weight to 4 large plastic bins, and the contents were mixed to ensure uniform distribution of the batches. The diet was placed in 22.7 kg (50 lb) aliquots in the plastic-lined bags in which the pellets were originally supplied, then excess air was expressed and the opening of the bag was sealed with plastic tape to provide an airtight closure. The control diet was prepared by using the same methods, except that no doxycycline was added to the oil and only 68 kg (150 lb) of diet was made. Both diets were prepared on the day before starting treatment. The bags of diet were stored at 20[degrees]C-24[degrees]C (68[degrees]F-76[degrees]F). As needed, the bags were transferred into a plastic garbage can with a tight-sealing lid inside the bird rooms. Diet was added to 4 stainless-steel bowls in each cage daily, which ensured that excess (unconsumed) diet and multiple feeding stations were always present. Each morning, diet left over from the previous day was discarded and new diet was provided.

Study design

Treatment and control diets were first offered on day 0 and fed through day 47. The treatment period was extended from the planned 45 days to 47 days to accommodate scheduling issues. Through day 50, the birds were observed at the same time each morning, and changes in attitude, activity level, and droppings were recorded. On days 3, 7, 14, 21, 28, 35, and 42, the rooms were entered when the lights first came on at 0830 hours and food was removed from the cages. Blood samples (0.6-0.9 mL) were then collected from the treatment group within a 13-20-minute time period, and the birds were also weighed and examined. The control group was then processed in the same manner as the treatment group. Blood samples from the treatment group were analyzed for doxycycline concentration. To assess possible adverse effects, packed cell volume, total plasma solids, and levels of selected plasma biochemical parameters (aspartate aminotransferase [AST], bile acids, creatine kinase [CK], uric acid, glucose, calcium, phosphorus, total protein, albumin, globulin, potassium, and sodium) were measured in both groups by using a commercial chemistry analyzer (Avian/Reptile Profile Plus reagent rotor used with Vetscan, Abaxis, Union City, CA, USA) 5 days before treatment and on days 21 and 42. An additional blood sample was collected 3 days after treatment ended (day 50) from a single bird in the control group that showed mild elevation of plasma AST and CK on days 21 and 42. To assess the impact of treatment on microbial gut flora, swabs were collected from the cloaca of all the birds and were used to inoculate blood agar, MacKonkey agar, and Sabouraud Dextrose agar plates at 2 days before the start and at 2 days after the end of the 47-day treatment period. The plates were examined specifically for the presence of the following potential pathogens: gram-negative bacteria, hemolytic gram-positive bacteria, and yeast. The birds were held for an additional 16 days after treatment, and, during that time, they were observed on 11 of 16 days, weighed on 4 of 16 days, and switched to a seed diet starting on posttreatment day 3. The birds were then returned to the aviculturist who reported that no problems were observed in the 3-month period after their return.

Measurement of plasma doxycycline concentration

Plasma doxycycline concentrations were determined by means of high-pressure liquid chromatography with ultraviolet detection by using a validated method described previously. (29) Pooled plasma samples from cockatiels that had not been treated with doxycycline or other drugs were used to prepare calibration curves and to check for interfering peaks. The limit of quantification was 0.10 [micro]g/mL.



Results of pretreatment tests for C psittaci DNA and aerobic bacterial and fungal cloacal pathogens were negative for the presence of pathogens. Doxycycline concentrations in the treatment group ranged from 0.98 to 3.83 [micro]g/mL with an overall median of 2.09 (Fig 1; Table 1). The total amounts of diet used for the 47-day trial were 29.8 kg (65.7 lb) of treatment diet and 36.9 kg (81.3 lb) of control diet. Note that much of this was unconsumed diet that was discarded daily.

No clinically important adverse effects associated with treatment were identified. Throughout the treatment period, the activity level, behavior, and droppings of the birds were normal. No aerobic bacterial pathogens or yeast were found in posttreatment cultures from the cloaca. The median body weight changes from the date of acquisition to the start of treatment (day 0) were +7.5% (range, -1.4% to +14.7%) and +3.1% (range, -0.9% to +13.0%) for the treatment and control groups, respectively. The median weight changes from treatment day 0 to day 42 (Fig 2) were +0.45% (range, 4.5% to +13.0%) and -1.3% (range, -8.5% to +5.7%) for the treatment and control groups, respectively. One bird in the treatment group showed a marked weight gain, which increased 7.5% during the pretreatment period and an additional 13.7% during treatment (total weight gain, 21.2%). At the end of treatment, this bird was considered markedly obese. After feeding this bird a commercial seed diet in the posttreatment period for 16 days, the total weight gain compared with the day of acquisition was reduced to 11.6% and the body condition score was considered slightly obese.

Packed cell volume, plasma total solids, and results of plasma biochemical analyses were within reference limits in both groups of birds before and during treatment, except for 1 bird in the control group that had increased AST activity (834, 738, and 601 U/L on days 21, 42, and 3 days after treatment, respectively; reference range, 107481 U/L). This bird also showed slightly increased CK activities (583 and 814 U/L on day 21 and 3 days after treatment, respectively; reference range, 69-523 U/L). (30)


We assumed that collecting blood samples for plasma doxycycline analysis after a 10-hour period of darkness would represent the lowest doxycycline concentrations during the 24-hour period (ie, trough samples) because the birds would likely eat less at night. Results of the present study show that cockatiels fed a specific pelleted diet that was medicated with 300 mg doxycycline hyclate per kilogram of diet maintained a trough plasma doxycycline concentration of median 2.09 [micro]g/mL (range, 0.98-3.83 [micro]g/mL). All but 1 measured plasma concentration during 42 days of treatment exceeded the target level of 1 [micro]g/mL. These concentrations were slightly lower than those maintained by feeding a seed diet medicated with 500 mg doxycycline per kilogram of feed (mean [SD], 3.19 [+ or -] 1.49 [micro]g/mL (23) or water medicated with 400 mg doxycycline/L (2.58 [+ or -] 1.00 [micro]g/mL). (7)

Both treatment and control diets were well accepted, and no adverse effects were noted, except for marked weight gain in 1 treatment group bird. This weight gain may have been due to individual bird characteristics or palatability of the diet. The only cockatiel that showed abnormal plasma biochemical values was a control group bird, and the mild increases in AST and CK activities were unexplained. Both birds were observed by the owner for 3 months after the study, and no abnormal behavior or other problems were reported.

Consistency in preparation of this medicated diet according to the methods listed is essential to successful treatment. Commercially available generic doxycycline capsules were used rather than bulk drug to assure potency. Pure soybean oil was used to create the doxycycline suspension. It is not known if other oils would be suitable because doxycycline is unstable when prepared in some compounded formulations. For example, doxycycline compounded for oral administration mixed with chocolate milk and other common household food and drinks for people was stable for only 7 days. (31) Our study showed that adequate doxycycline concentrations were maintained for at least 42 days by using a medicated diet prepared on the day before administration and stored at room temperature. Future experiments should determine the stability of doxycycline in medicated pellets to determine if the premade diet can be stored.

Preparation of this medicated food is within the capabilities of a skilled veterinary staff. A cement mixer was used to prepare the large quantity of doxycycline-medicated pellets produced for this study. A smaller batch of pellets probably could be prepared by using a smaller tumbling mixer, such as the type used by hobbyists to tumble stones; however, this was not tested. Uniform mixing of a doxycycline-oil mixture, complete absorption of the doxycycline-medicated oil, and maintaining an intact pellet are important parameters to assess if an alternative mixing regimen is used.


Doxycycline is most often used in avian medicine to treat infection caused by C psittaci. As noted above, there is no proven relationship between doxycycline plasma concentrations and treatment efficacy because reported therapeutic trials in naturally infected cockatiels are lacking. In addition, treatment efficacy cannot be based on in vitro susceptibility testing because results have been method-dependent, and it is not known if in vitro results predict in vivo activity. (32-35) Historically, successful treatment regimens in psittacine birds have been designed to maintain trough doxycycline concentrations above 1 [micro]g/mL for 45 days, and this has become the standard recommendation for treatment. (11) The medicated diet used in our study safely maintained trough doxycycline plasma concentrations above 1 [micro]g/mL for the 42 days that concentrations were measured and should be effective for treatment of most C psittaci infections in cockatiels. However, because ultimate clearance of C psittaci from the host likely depends on host immunity in addition to antimicrobial drug levels, no treatment regimen based on a pharmacologic standard can be guaranteed to eliminate infection in every bird. A shorter duration of treatment possibly can be effective. (28)

Doxycycline is also used to treat spiral bacterial infections in cockatiels. (6,7) Spiral bacteria have been associated with weight loss, pharyngeal inflammation, and upper respiratory disease signs. (6,7) The medicated diet used in our study may be effective for treatment because plasma concentrations were similar to those achieved in a successful clinical trial when using doxycycline-medicated water for a treatment period of 21 days. (7)

Judicious use of doxycycline can successfully control avian chlamydiosis and spiral bacterial infection; however, this drug must be used responsibly. Doxycycline resistance has been reported for a related organism, Chlamydia suis, possibly related to plasmid transfer from a different species of bacteria and widespread use of tetracyclines in swine. (36-39) Indiscriminate use of doxycycline-medicated feed could potentially lead to antimicrobial resistance in C psittaci or other bacteria carried by treated birds. Doxycycline should only be used when there is clear evidence of a need to treat the birds for C psittaci, spiral bacterial infection, or infection with a bacterium known to be susceptible to doxycycline. Evidence should include a confirmed diagnosis or direct association with an infected bird. Neither this, nor any other doxycycline treatment regimen should be used to indiscriminately treat uninfected or unexposed birds.

Limitations of this study should be considered before extrapolating the results to field conditions. Factors that impact food consumption could alter the systemic drug concentrations that are achieved with medicated feed. The cockatiels used in our study were healthy, adult, nonbreeding birds that were housed indoors under controlled conditions. Along with other food types, these birds had been offered a pelleted diet during weaning, which may increase recognition of the pellets as food. We also increased the likelihood that our experimental birds would accept the medicated pelleted diet by gradually converting the birds from a seed to a nonmedicated pelleted diet over a 10-day period before starting treatment. Some cockatiels may be reluctant to accept novel foods and may be difficult to switch from a seed to a pelleted diet. If this medicated diet is offered abruptly to cockatiels that have not been conditioned to eat a pelleted diet, then food consumption and bird body weight should be monitored because consumption of the diet may be less than shown by the birds in this trial. It is also essential to use only the pelleted diet specified in this experiment because pelleted diets vary in palatability. In addition, the base diet used in our study contained reduced fat, which was increased when the soybean oil-doxycycline mixture was added to the pellet. Adding the soybean oil-doxycycline mixture to a different pellet formulation may result in excess dietary fat.

Other factors that alter food consumption could affect the plasma doxycycline concentrations achieved by feeding a doxycycline-containing pelleted diet. For example, birds that are reproductively active or exposed to cold weather might increase their intake of a medicated diet. In contrast, sick birds might consume less. In addition, cockatiels should not be allowed access to dietary sources of calcium (eg, cuttlebone, mineral block, or grit) during treatment with doxycycline to avoid potential chelation of doxycycline in the gastrointestinal tract. In summary, feeding a pelleted diet with doxycycline added at 300 mg per kilogram of diet may be suitable for treating C psittaci and spiral bacterial infections in cockatiels that will consume a pelleted diet.

Acknowledgments: We thank Josh Maple of Maple Leaf Aviaries, Garner, NC, USA, for the loan of the birds. This study was funded by Roudybush [TM] and the State of North Carolina.


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Keven Flammer, DVM, Dipl ABVP (Avian), J. Gregory Massey, DVM, Dipl ABVP (Avian), Tom Roudybush, MS, Caroline J. Meek, and Mark (3. Papich, DVM, MS, Dipl ACVCP

From the Departments of Clinical Sciences (Hammer, Massey, Meek) and Molecular Biosciences (Papich), College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough St, Raleigh, NC 27606, USA; and Roudybush, 340 Hanson Way, Woodland, CA 95776, USA (Roudybush).
Table 1. Plasma concentrations of doxycycline in
cockatiels (n = 9) fed a pelleted diet that contained 300
mg doxycycline hyclate per kilogram of feed.

                   Plasma doxycycline

Day of treatment   Median     Range

3                   2.20    1.24-2.93
7                   2.71    1.42-3.58
14                  2.17    0.98-3.78
21                  1.91    1.39-3.83
28                  2.19    1.60-3.28
35                  2.31    1.32-3.24
42                  1.65    1.20-3.65
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Author:Flammer, Keven; Massey, J. Gregory; Roudybush, Tom; Meek, Caroline J.; Papich, Mark G.
Publication:Journal of Avian Medicine and Surgery
Article Type:Report
Date:Sep 1, 2013
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