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Mycobacteriosis is an avian disease that is most commonly caused by Mycobacterium avium or Mycobacterium genavense. In order to optimize molecular laboratory tests for diagnosing mycobacteriosis in birds, we compared four methods of rapid DNA extraction with isolates of M. avium, M. genavense, and Mycobacterium fortuitum. DNA extraction methods included enzymatic lysis, boiling for 30 min followed by enzymatic lysis, four cycles of freezing and thawing followed by enzymatic lysis, and bead beating followed by enzymatic lysis. The DNA yield and purity for the four methods were evaluated by spectrophotometry and compared. The bead beating with enzymatic lysis technique yielded significantly purer and higher concentrations of extracted DNA compared with other DNA extraction methods. All four methods yielded extraction products for all three organisms that were successfully amplified by polymerase chain reaction (PCR) for a fragment in the 65-kD heat shock protein gene. Subjectively, the PCR amplification products were most abundant for samples extracted by bead beating with enzymatic lysis.

A long-acting, biodegradable, controlled-release formulation of oxytetracycline (CR-OTC) was evaluated in 18 adult Japanese quail (Coturnix coturnix japonica) following a single subcutaneous (s.c.) injection. Prior to characterizing the release of oxytetracycline (OTC) from the CR-OTC, the pharmacokinetic parameters of intravenously (i.v.) administered OTC were determined. Concentrations of free OTC were measured using a bioassay. The plasma concentration-time profile of OTC after a single i.v. injection at 20 mg/kg was best fit to an open two-compartmental model, with the following pharmacokinetic parameters: area under the curve (AUC) = 36.72 mg. h/L, terminal elimination half-life = 2.34 h, clearance (Cl) = 0.545 L/kg/h. Plasma [OTC] was >1.0 micro g/mL for at least 4 h following i.v. injection. The CR-OTC gel was well tolerated at a dosage of 1500 mg/kg s.c. Plasma [OTC] rose to >1.0 micro g/mL within 24 h; it remained >1.0 micro g/mL for at least 10 days in all birds sampled at that time point (n = 9) and for at least 18 days in two of nine birds. Using a deconvolution technique, it was determined that approximately 54.8% of the administered OTC was released from the CR-OTC over the 45-day observation period. This long-acting, biodegradable controlled-release OTC formulation may have potential for the treatment of chlamydophila infections and other OTC-sensitive bacteria in Japanese quail, however further studies are necessary to determine its safety and clinical application.

In this study we compared culture, acid-fast stains, and polymerase chain reaction (PCR) for the detection of acid-fast organisms in fecal and tissue samples from Japanese quail (Coturnix coturnix japonica) that were experimentally inoculated intravenously with Mycobacterium avium. For culture, three different culture media (modified Herrold egg yolk with mycobactin; Lowenstein-Jensen [L-J]; and L-J with cyclohexamide, naladixic acid, and lincomycin) were tested to determine which medium had the greatest success in isolating mycobacteria. Acid-fast staining methods included Zichl-Neelsen (Z-N) and Truant. The PCR assay detected mycobacterial DNA with primers specific for the 65-kD heat shock protein gene. Culture was considered the "gold standard." Compared with other culture media, L-J yielded more positive cultures and greater numbers of colonies on positive tubes, and incubation times were shorter. Mycobacterium avium was isolated from all of the harvested tissue samples (liver, spleen, and intestine) of inoculated birds. Mycobacteria were isolated from 53% (69/130) of fecal samples from inoculated birds. As the disease advanced, fecal culture was positive on more culture days, indicating that the culture-positive rate was higher later in the course of the disease. Compared with culture, all of the laboratory methods had 100% specificity for the tissue samples. Sensitivities for the tissue samples were 82.6% (Z-N), 95.7% (Truant), and 100% (PCR). For the fecal samples, the specificity was95% for all methods. Sensitivities compared with fecal culture were 7.2% (Z-N), 30.4% (Truant), and 20.3% (PCR). Tissue and fecal samples from the two control birds were negative for acid-fast organisms by any method. These results were comparable with clinical cases of avian mycobacteriosis where culture and PCR of tissue samples seem to be the most sensitive and specific laboratory tests and evaluation of fecal samples still remains challenging. On the basis of the results of this study, identification of mycobacteria in fecal samples from Japanese quail can be optimized by repeated cultures and Truant acid-fast staining of fecal smears.

Enrofloxacin is a fluoroquinolone antibiotic that is effective against many of the common gram-negative bacterial pathogens in raptors. This 3-period crossover study was designed to compare the pharmacokinetics of enrofloxacin after oral, intramuscular, or intravenous administration in red-tailed hawks (Buteo jamaicensis; n = 8) and great horned owls (Bubo virginianus; n = 5). In each study period, birds received a single dose of an injectable formulation of enrofloxacin (15 mg/kg) by 1 of these 3 routes. Oral administration was accomplished by force-feeding each bird a small freshly killed mouse that had received an intraperitoneal injection of enrofloxacin (in-prey). Intramuscular injections were divided into 2 sites in the pectoral musculature. Serial plasma samples were taken before administration and at 0.25, 0.5, 1, 2, 4, 8, 12, 24, and 48 hours after administration. Oral administration resulted in plasma concentrations of enrofloxacin that peaked at 4–8 hours in both species. Plasma concen...

This study characterized and compared the pharmacokinetics of piperacillin after single 100 mg/kg i.m. injections in nine red-tailed hawks (Buteo jamaicensis) and five great horned owls (Bubo virginianus) over 48 hr by a modified agar well diffusion microbial inhibition assay. The mean maximum plasma piperacillin concentrations were 204 microg/ml and 221 microg/ml for the hawks and owls, respectively, and times of maximum concentrations were 15 min and 30 min, respectively. The calculated mean terminal elimination half-lives were 77 min in the hawks and 118 min in the owls. Area-under-the-curve values were 218 +/- 52 microg x hr/ml in the hawks and 444 +/- 104 microg x hr/ml in the owls. On the basis of the most common minimal inhibitory concentration (90%) for various bacterial isolates from clinical samples of 8 microg/ml, analysis of the data suggests that the maximum dosing interval for piperacillin at 100 mg/kg in medium sized raptors should be 4-6 hr.

Diagnosis of avian mycobacteriosis, caused by Mycobacterium genavense or species belonging to the Mycobacterium avium complex (MAC), is problematic. Polymerase chain reaction (PCR) offers rapid and sensitive detection of minute quantities of DNA, and conventional protocols have been used for evaluating avian specimens. The recent development of real-time PCR offers several advantages over conventional PCR. In attempts to improve diagnosing avian mycobacteriosis, a real-time TaqMan PCR assay was developed targeting the 65-kD heat shock protein gene of M. genavense and MAC spp. Nineteen reference isolates, 16 clinical isolates, and 32 avian tissue samples were used to evaluate the assay. When sufficient amplicons were produced, the species of mycobacteria was determined by standard sequencing of TaqMan PCR products and compared with results from commercial mycobacteriology laboratories and/or standard sequencing of conventional PCR products. The TaqMan PCR detected DNA from reference isolates of M. genavense, MAC spp., and Mycobacterium tuberculosis complex spp. Of the clinical isolates, the TaqMan PCR detected DNA from 10 of 12 Mycobacterium avium avium isolates and two of three Mycobacterium avium intracellulare isolates. For the tissue samples, the TaqMan PCR amplified DNA in six of nine samples that were identified by sequencing of conventional PCR products and/or by commercial mycobacteriology laboratories as being MAC spp. positive and three of four samples that were positive for M. genavense. There was some disagreement between speciation results from the TaqMan PCR and those from commercial mycobacteriology laboratories or conventional PCR or both. This disagreement was suspected to be because of relatively small numbers of base pairs in the TaqMan PCR products. The TaqMan PCR may provide a useful tool for evaluating clinical samples for DNA from mycobacteria species that most commonly infect birds; however, further refinement is needed in order to improve sensitivity and provide more accurate speciation.

Mycobacterial infections are an important cause of morbidity and mortality in birds and a considerable diagnostic challenge until the disease is advanced. In order to develop more clinically useful antemortem tests, a biological model was created that replicated naturally occurring disease. Japanese quail (Coturnix coturnix japonica; n = 8) were inoculated intravenously with Mycobacterium avium. Two additional birds served as uninoculated controls. Mean survival time of the inoculated birds was 68 +/- 13 days postinoculation (PI). Seven of the eight inoculated birds died naturally. Clinical and postmortem abnormalities in inoculated birds were characteristic of naturally occurring mycobacteriosis. Abnormal clinical findings included decreased activity, feather erection, and sudden death. Mean body weight and packed cell volume declined and mean total white blood cells (primarily heterophils, bands, and monocytes) increased from 28 days PI onward. Similar to birds that are naturally infected with mycobacteriosis, the inoculated birds were thin and had severe hepatosplenomegaly on postmortem examination. All eight birds had lesions in the liver, spleen, intestine, lung, gonads, and serosa. Less commonly affected tissues included bone marrow, thymus, gizzard, heart, pancreas, and brain. Lesions were invariably severe in the liver and spleen. These gross postmortem findings were consistent with natural infections of avian mycobacteriosis. Mycobacterium avium was isolated from the liver, spleen, and intestine of all inoculated birds. Both control birds remained disease free and culture negative. This inoculation protocol is a reliable and practical means of inducing avian mycobacteriosis for further study.

The pharmacokinetics of ceftiofur sodium were determined in domestic chicks, turkey poults, adult cockatiels (Nymphicus hollandicus), and adult orange-winged Amazon parrots (Amazona amazonica) after subcutaneous (chicks and turkey poults and intramuscular (i.m.) dosing (cockatiels and Amazon parrots). Turkey poult data were best fit to a single exponential model with disappearance half-lives (t1/2) of 8.6, 7.4 and 5.6 h after doses of 0.12, 0.24 and 0.48 mg ceftiofur free acid equivalents (CFAE)/poult, respectively. Data from chicks were best fit to a biexponential model with primary and secondary half-lives of 2.2 and 7.5, 3.7 and 6.8, and 3.8 and 5.3 h after doses of 0.04, 0.08 and 0.16 mg CFAE/chick, respectively. Cockatiel and Amazon parrot data were best fit to a biexponential model with primary and secondary half-lives of 0.28 and 2.5, and 0.93 and 7.9 h, respectively, after doses of 10 mg CFAE/kg body weight. The maximum concentration (Cmax) and area under the concentration time curve (AUC) in chicks and poults were dose-proportional. The Cmax for cockatiels was 5.2 micrograms/mL and for Amazon parrots was 11 micrograms/mL. Clearance in cockatiels and Amazon parrots were 11.3 and 3.8 mL/min/kg, respectively, and reflected the much greater AUC seen in Amazon parrots. Clearance values of ceftiofur were similar in chicks and Amazon parrots, slightly greater in turkey poults and greatest in cockatiels. These results indicate that pharmacokinetic differences must be considered when establishing dosage regimens for different avian species.