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Evaluation of multidrug therapy with azithromycin, rifampin, and ethambutol for the treatment of mycobacterium avium subsp avium in ring-neck doves (Streptopelia risoria): an uncontrolled clinical study.

Abstract: An uncontrolled clinical study was conducted to evaluate the efficacy of a multidrug protocol in 16 ring-neck doves (Streptopelia risoria) from a flock naturally infected with Mycobacterium avium subsp avium. The doves were considered infected on the basis of a high prevalence of infection in a group of 29 birds that were euthanatized from the same flock and clinical signs and pathologic results consistent with infection in the remaining birds. All birds were treated with azithromycin (43 mg/kg), rifampin (45 mg/kg), and ethambutol (30 mg/kg) administered orally once daily for 180 days. Five birds died during treatment and were confirmed positive for mycobacteriosis on postmortem examination. Of the remaining 11 birds, infection and disease were present in 9 (81.8%) at the end of the treatment. Postmortem investigation showed that 2 mycobacterial isolates were resistant to ethambutol, intermediately sensitive to rifampin, and sensitive to azithromycin. Microscopic examination of liver sections equivalent of those that would be taken for biopsy showed that biopsy as a method of monitoring birds for treatment success had poor sensitivity. Toxicity associated with drug therapy was not observed in these doves nor in 6 outwardly healthy ring-neck doves exposed to the same treatment. The results of this study showed that this protocol of azithromycin, rifampin, and ethambutol has poor efficacy when administered for 180 days for treatment of doves infected with M avium subsp avium.

Key words: mycobacteriosis, Mycobacterium avium subsp avium, treatment, ethambutol, rifampin, azithromycin, ring-neck doves, Streptopelia risoria

Introduction

Mycobacteriosis is a common disease of companion birds. It is caused by a range of mycobacteria, including, but not limited to, Mycobacterium avium subsp avium, M intracellulare, M genavense, M tuberculosis, M gordonae, M fortuitum, M simiae, and M celatum. (1-10) There has been considerable discussion in the literature as to whether birds with mycobacteriosis should be treated or euthanatized. (11-15) The arguments for euthanasia are the zoonotic potential of many of these organisms, the risk of transmitting the disease to other birds, and the need for an expensive and prolonged course of multidrug therapy. Difficulty of drug administration to some species of birds, antimicrobial drug resistance in many isolates, and poor owner compliance also make a successful course of treatment challenging. (13,16-20)

Despite these controversial opinions, very few reports of attempts to treat birds affected by mycobacteriosis have been published. Such reports usually involve only a limited number of birds and consist mainly of individual case reports or case series, and rarely is the organism being treated characterized or its susceptibility to the antibiotics used known. (9,14,15,21) A wide range of antibiotics, dosages, and lengths of therapy have been recommended with variable or incompletely known outcomes. (9,13-15,21) Most of these regimens have been extrapolated from the human pediatric literature and calculated by metabolic allometric scaling or based on anecdotal reports. (13,14,21,22) Additionally, no studies as yet have evaluated the potential adverse effects that multiple-drug treatment protocols might have in birds. This is important because most antimycobacterial drugs have adverse effects. For example, rifampicins may induce hepatic microsomal enzymes, ethambutol has been associated with the development of optic neuritis, and macrolides may cause diarrhea. (18,23)

Mycobacterium avium subsp hominissuis infections commonly occur in people experiencing advanced stages of AIDS. (24,25) Treatment regimens with one of the new macrolide antibiotics, azithromycin or clarithromycin, in combination with ethambutol and one of the rifampicins (rifampin or rifabutin) appear to be more effective in these patients than older combinations of drugs. (2,18) If true, this combination of drugs may also be effective in birds infected with M avium complex. Similar protocols have been recommended for birds in the past (9,12,14,21) and are likely to be effective against mycobacterial species known to infect birds. (4,26)

We report the results of an uncontrolled clinical study undertaken to determine whether a recommended multidrug treatment regimen consisting of azithromycin, ethambutol, and rifampin administered orally (4,9,12,14,21,26) during a period of 6 months will eliminate infection from ring-neck doves (Streptopelia risoria) naturally infected with M avium subsp avium. We also sought to determine whether this treatment protocol would be free of adverse effects, if the mycobacteria would develop antibiotic resistance, and if a liver biopsy at the end of treatment, as has been previously reported, (14,27) could be used to assess treatment success.

Materials and Methods

Specimens

Seventy adult ring-neck doves were obtained from an aviary near Hillsboro, TX, USA, in July 2005. These birds came from a flock where more than 60 doves had died during the previous 6 months. Mycobacterial infection had been previously confirmed in these birds at the Texas Veterinary Medical Diagnostic Laboratory (College Station, TX, USA) and the condition of some of the surviving birds (decreased productivity, weight loss, depression) was consistent with a chronic infectious disease such as mycobacteriosis affecting the flock. All birds were housed in a small shed that was open to and surrounded on 2 sides by an outdoor flight cage. Heavy fecal contamination was present on the floor and perching surfaces of the shed, and food and water were contaminated with feces. The surviving birds were donated and transferred to an isolation building at the College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA. The disease, etiology, differential susceptibility to disease, and efficacy of different diagnostic methods was investigated in 29 of these birds and was reported previously. (28-30)

Of the surviving group of 70 ring-neck doves, 16 were randomly selected to be included in an uncontrolled clinical study. Treatment was begun in these 16 doves immediately after being received at the Schubot Exotic Bird Health Center. Infection status was based on the high prevalence of mycobacterial infection (62%) in a previously euthanatized group of 29 doves from the same flock. (28) Additionally, Western blot analysis of plasma samples and hematologic testing and plasma protein electrophoresis results were used as criteria for preliminary antemortem classification as infected. (29-31) This included a combination of 3 or more of the following clinicopathologic results consistent with mycobacteriosis in doves (28-31): positive results of Western blot analysis, anemia, leukocytosis, heterophilia, monocytosis, hyperglobulinemia, and hypoalbuminemia. All 16 doves in the trial met the established criteria for antemortem diagnosis of mycobacterial infection.

As defined by Friedman et al, (32) an uncontrolled clinical trial does not include control groups. We considered withholding treatment from infected doves for 6 months was unethical given that discomfort, overt poor quality of life, and death are outcomes of untreated systemic avian mycobacteriosis. Therefore, infected untreated birds were not used as controls during this study. However, to investigate the potential adverse effects of these drugs, the same treatment was administered to 6 apparently healthy ring-neck doves that had been housed at the Schubot Exotic Bird Health Center aviary for the previous 6 years. These birds were considered free of mycobacterial infection because they were serologically negative by Western blot analysis, demonstrated no clinical signs of mycobacteriosis, and had normal hematologic and biochemical parameters.

This research was approved by the University Laboratory Animal Care Committee at Texas A&M University (animal use protocol 2005-56).

Treatment protocol

The 16 doves from the infected flock were housed in suspended wire cages in 2 groups of 5 and 1 group of 6 birds per cage in a biological level 3 isolation building. Birds were identified by a numbered band placed around the tarso metatarsus. Water and mixed seeds were offered ad libitum. Birds were administered a combination of azithromycin (43 mg/ kg), rifampin (45 mg/kg), and ethambutol (30 mg/kg) PO once a day for 180 days following recommended treatment regimens. (21) Suspensions of each medication in simple syrup were prepared daily and administered immediately to each bird by gavage. Birds were examined daily when they were handled for treatment. Birds that developed severe weight loss or showed signs of lethargy, stupor or coma, severe dyspnea, pain, or prolonged anorexia during the treatment period were euthanatized.

Treatment evaluation

At the end of the treatment period, all surviving doves were anesthetized with an intramuscular injection of ketamine/xylazine and euthanatized by exsanguination by cardiocentesis. Postmortem examination was done within 4 hours of euthanasia. Representative sections of liver, spleen, lung, and bone marrow were collected for polymerase chain reaction (PCR) assay and mycobacterial culture. Representative sections of liver, spleen, heart, lungs, air sacs, kidneys, intestines, gonads, adrenal glands, thyroid gland, trachea, esophagus, ventricle, proventriculus, crop, skeletal muscle, and bone marrow were fixed in 10% buffered formalin and stained with hematoxylin and eosin and Ziehl-Neelsen stain. Birds were considered cured if no macroscopic or microscopic lesions consistent with active mycobacteriosis were identified in any organ; the liver, spleen, and bone marrow were culture and PCR negative; and acid-fast organisms were not seen in Ziehl-Neelsen-stained sections in any organ.

Immediately after euthanasia in surviving doves and before postmortem examination in all doves, liver and bone marrow were collected by the biopsy technique used in live birds. Bone marrow samples were obtained by aspirate from the distal ulna with a 20-gauge needle and 6-mL syringe and frozen at -80[degrees]C. (29) The coelomic cavity was opened aseptically by a ventral midline approach, and a liver section approximately 4 x 4 x 4 mm was excised from the caudal border of the right lobe. (29) This is the location and size of tissue that would be collected from a live bird of this size during a routine liver biopsy. A small portion was saved for PCR assay and culture, and the remaining tissue was formalin-fixed for histologic examination.

Microscopic lesions

Microscopically, inflammatory lesions were scored semiquantitatively as mild, moderate, or severe based on the degree of severity and extent of the observed lesions. (28,29) The numbers of acid-fast bacilli were graded relatively as none, few, many, or massive. (28,29) Congo red staining was used to investigate the presence of amyloid in liver and spleen. (28,29)

Identification of mycobacteria in tissues

Swabs from macerated liver, spleen, lung, and bone marrow from all the doves investigated were inoculated into 5 mL of Middlebrook 7H9 broth (Becton Dickinson, Franklin Lakes, NJ, USA) containing 0.5% (vol/vol) glycerol and 10% (vol/ vol) oleic acid albumin and incubated at 39[degrees]C. (33) Cultures were inspected weekly for microbial growth and examined for the presence of mycobacteria by Ziehl-Neelsen staining.

DNA was extracted from all investigated tissues and isolates with the Puregene Genomic DNA Purification Kit (Gentra Systems, Minneapolis, MN, USA) following manufacturer instructions. PCR screening for mycobacterial DNA was performed with primers T1 [5'GGGTGACGCG(G/A)CATGGCCCA-3'] and T2 [5'-CGGGTTTCGTCGTACTCCTT-3'] for amplification of the 236-bp dnaJ gene as described. (34) The PCR reaction parameters were as follows: 1 initial cycle of 94[degrees]C for 5 minutes; 40 cycles at 94[degrees]C for 30 seconds, 60[degrees]C for 45 seconds, and 72[degrees]C for 1 minute; and an additional elongation step at 72[degrees]C for 5 minutes. Positive (M avium) and negative reaction control (DNA-RNA-free sterile water) were used in each reaction. Amplified DNA was visualized after electrophoresis on a 1.5% ethidium bromidestained agarose gel. The PCR products were purified with QIAquick PCR Purification Kit (Qiagen Inc, Valencia, CA, USA). Sequencing reactions were performed with an ABI Prism Big Dye Terminator v3.0 Cycle Sequencing Kit (Applied Biosystems Inc, Foster City, CA, USA). Nucleotide sequences were determined with an ABB 100 automated DNA sequencer (Applied Biosystems). All sequences were aligned with Clustal X 1.8l (35) and were compared with sequences retrieved from GenBank (www.ncbi. nlm.nih.gov/Genbank/index.html).

Investigation of adverse effects

The potential adverse effects of these drugs were investigated in 6 outwardly healthy doves housed at the Schubot Exotic Bird Health Center aviary. These birds were treated and monitored in the same way as the naturally infected birds. Blood samples were collected from the basilic vein at days 0, 60, 120, and 180. Blood smears were made immediately; the remaining blood was refrigerated, and the packed cell volume (PCV) was measured within 2 hours of collection. Total protein, albumin, globulin, and uric acid concentrations, and aspartate aminotransferase (AST) and creatinine kinase (CK) activities were also determined with an Ortho Vitros 250 chemistry analyzer (Block Scientifics, Holbrook, NY, USA) at the Clinical Pathology Laboratory, Department of Pathobiology, College of Veterinary Medicine, Texas A&M University. Packed cell volume was evaluated after samples in hematocrit tubes were centrifuged for 5 minutes. Estimated white blood cell counts were performed. (29) Plasma protein electrophoresis was performed with the Titan Gel electrophoresis kit (Helena Laboratory, Beaumont, TX, USA) following the manufacturer's instructions.

Response to therapy in healthy doves was monitored by comparing changes in the hemogram, PCV, plasma protein and uric acid concentrations, AST and CK activities, and monthly weight. Increases in AST and CK plasmatic activity levels and uric acid plasma concentrations, changes in plasma protein values, the onset of gastrointestinal signs (diarrhea, anorexia, vomiting, and weight loss), or the presence of histopathologic evidence of optic neuritis were considered signs of adverse effects or drug toxicity. After 180 days of treatment, the healthy birds were euthanatized, and samples were collected and further investigated for mycobacteriosis as previously described for the infected birds.

Because clinical signs associated with mycobacterial disease and those associated with adverse drug effects are similar, evidence of drug toxicity in infected birds was assessed by histopathologic evidence of optic neuritis only.

Antimicrobial sensitivity testing

Liver and spleen tissue samples from 4 untreated doves from the same flock (28) and 4 treated doves that were culture positive at the end of the trial were submitted to the Mycobacteriology Laboratory at the National Jewish Medical and Research Center, Denver, CO, USA, for additional identification through high-performance liquid chromatography and subsequent antimicrobial sensitivity testing by radiometric minimal inhibitory concentration determination in liquid medium (7H12 broth, Bactec-460, Becton Dickinson, Franklin Lakes, NJ, USA).

Statistical analysis

Hematologic tests, biochemical analysis, and plasma protein electrophoresis values from the 6 healthy doves were compared between sample dates and analyzed by a 2-tailed Student's 1 test for parametric data and a Mann-Whitney U test for nonparametric data. Test statistics were considered significant at P < .05. All statistical analysis was conducted with the formula package in Prism 5 for Windows (GraphPad Software Inc, available on-line at www.graphpad.com).

Results

Survival

Five of the 16 birds (31.3%) considered infected died during treatment. The first bird died after 21 days of treatment. It was in a poor condition and severely underweight and anemic when treatment started. On postmortem examination, infection status was confirmed by the presence of granulomatous hepatitis, splenitis, enteritis, and pneumonia. Massive numbers of acid-fast organisms were observed in these lesions.

A second bird was found moribund lying on its breast with severe dyspnea at day 120 and was euthanatized. An additional bird died spontaneously the same day. In both cases, death was attributed to mycobacteriosis based on the presence of severe granulomatous inflammatory lesions in the liver, spleen, and lungs and the identification of acid-fast organisms.

The last 2 birds died within the week before the end of the treatment. One was a sexually active male (testes 10 mm X 5 mm) in good physical condition, including abundant abdominal and coronary groove fat; the other dove was in fair physical condition but with an obvious distended abdomen. Hepatomegaly was present in both birds, and the ventral surfaces of the livers were surrounded by a gelatinous, serosanguineous exudate. Microscopic examination of liver and spleen confirmed mycobacteriosis in both birds based on the presence of granulomatous inflammation, moderate to massive numbers of acid-fast organisms, positive culture results, and results of PCR testing.

Of the 11 birds that survived treatment, 6 were male and 5 were female, all with active gonads. All but 2 birds had good pectoral muscle condition and were alert, eating well, and responsive to handling and oral administration of the drugs during all the treatment period.

Infection status in surviving birds

Mycobacteria were identified on postmortem examination in 9 of the 11 birds infected that completed the 180 days of treatment. A combination of Ziehl-Neelsen staining of selected tissues, mycobacterial culture, and PCR testing of liver, spleen, bone marrow, and lung samples was necessary to detect the mycobacteria. Ziehl-Neelsen staining of large histologic sections of tissues was the most sensitive technique and identified 9 infected birds (81.8% of samples). Sensitivity of each technique for those birds with confirmed mycobacterial infection at the end of treatment is shown (Table 1). No single organ or technique consistently yielded positive identification of mycobacteria. Only 2 of these birds were considered free of infection at the end of treatment based on Ziehl-Neelsen staining, culture results, and PCR testing. However, mild, multifocal, granulomatous inflammation was observed in the liver and spleen of these birds.

The sequences of the amplified dnaJ gene for isolates obtained after treatment were identical to the sequences of previously identified isolates in confirmed infected doves (28) and had 100% identity with the sequevar of M avium subsp avium that contains serotypes 2, 3, 4, and 9. (34)

Gross and microscopic lesions

Mild gross lesions characteristic of mycobacteriosis were observed in 9 of the 11 surviving birds (81.8%) initially considered infected. The most common organ affected was the spleen. Two birds had a severely enlarged spleen with multiple coalescing yellow nodules. Otherwise, the size of the spleen in the remaining birds was only 2 to 3 times the size of this organ in a normal bird, with very small yellow miliary foci embedded in the parenchyma.

Hepatomegaly (3 birds), discoloration of the liver (5 birds), and presence of small yellow foci within the liver (1 bird) were observed. Two birds had liver of normal appearance. A single yellow caseous nodule was observed in the lung of 1 bird, and another bird had small yellow nodules in an air sac. Kidneys were pale in 3 birds and enlarged and edematous in 2. Other isolated lesions included a small yellow nodule in the serosal surface of the ventricle and a subcutaneous granuloma of the neck. A single bird had a large ulcerated subcutaneous granuloma on the top of the head and another smaller mass within the left infraorbital sinus. These lesions had been recognized before the onset of treatment and did not regress during treatment. Gross lesions attributable to mycobacteria were not observed in the pancreas, intestines, brain, cerebellum, gonads, trachea, esophagus, proventriculus, or bone marrow.

Two histopathologic patterns were associated with persistent mycobacterial infections in the infected birds. The first was characterized by the presence of focal/multifocal granulomatous inflammation, consisting of a well-defined focus of inflammatory cells, sometimes surrounded by a ring of fibrous tissue, with or without central caseous necrosis. The second was characterized by diffuse granulomatous inflammation, with a variable degree of diffuse inflammatory cell infiltration without formation of discrete foci or nodules, absence of a fibrous capsule, and little or no caseous necrosis. Microscopic lesions were observed principally in the liver, spleen, and lung and sporadically in the bone marrow and ovary. Liver lesions were observed in all the birds. Both diffuse and multifocal hepatitis and splenitis were observed. Focal or multifocal lesions were considered mild to moderate, whereas in those birds with a diffuse pattern, moderate or severe inflammation predominated. A combined pattern of diffuse and multifocal granulomatous inflammation was observed in 1 liver. Amyloid deposits were identified in the livers of 4 birds with diffuse inflammation. Classic granulomas with histiocytes or multinucleated giant cells and fibrosis, surrounding a central core of necrosis, were prominent in 4 spleens (Fig la), 2 livers, 3 lungs, and 1 ovary. Scattered mild lymphoplasmacytic foci, some containing heterophils, were found in the liver of all birds, including the 6 healthy doves used for investigating safety of the treatment protocol. The cause of these lesions remains unknown but was not believed to be the result of a mycobacterial infection.

Acid-fast organisms (few to massive numbers) were usually completely restricted to the central core of necrosis observed in the granulomatous foci of the spleen (Fig 1 b), liver, lungs, and ovary. Other sections of these tissues, including those foci without caseous central cores or with diffuse inflammation, usually lacked detectable organisms.

Efficacy of fiver biopsy

Acid-fast organisms were only identified in 1 bird in liver biopsy samples. The liver biopsy samples of 2 additional birds were culture positive. The liver biopsy samples for all of the other birds were negative for mycobacteria for all the techniques. Overall, only 3 of 16 birds were positive by liver biopsy. All 3 positive birds were treated for 180 days.

Optic nerve examination in infected doves

Ten doves that received treatment were investigated for the presence of inflammation or demyelinization of the optic nerve, lesions consistent with ethambutol toxicosis. No histopathologic changes were detected in any of them.

Antibiotic susceptibility

Liver and spleen tissue samples from 4 untreated doves and 4 treated doves were sent to the National Jewish Medical and Research Center. After approximately 2 months, mycobacteria were grown from only 2 of the 8 samples submitted. One sample was obtained from a dove that did not receive treatment, and the other sample was obtained from a treated dove. Both isolates were identified as M avium by high-performance liquid chromatography, and both were susceptible to azithromycin, resistant to ethambutol, and showed intermediate resistance to rifampin.

Effect of treatment on healthy doves

All 6 healthy doves housed at the Schubot Exotic Bird Health Center Aviary survived the duration of the treatment. At postmortem examination, their physical condition was considered excellent. Gross postmortem examination and microscopic findings were unremarkable except for the previously mentioned mild foci of inflammation in the livers and for a localized airsacculitis detected in 1 bird, associated with the presence of a small metallic foreign body that had probably been inhaled. Acid-fast organisms were not observed in Ziehl-Neelsen-stained sections. Mycobacteria were neither cultured nor detected by PCR from either the liver or the spleen. No gross or microscopic lesions were observed in the optic nerve of these birds.

The mean weight of these 6 healthy birds increased slightly from the beginning of treatment to the end, although differences in monthly mean weights were not statistically significant. No significant changes were observed in PCV and plasma protein concentrations during the course of treatment. Changes in mean plasma AST and CK activities and uric acid concentrations were also not significant.

Discussion

This uncontrolled clinical study was designed to determine the efficacy and safety of a triple antibiotic treatment protocol of 6 months duration in a large number of ring-neck doves from a flock affected with spontaneous mycobacteriosis caused by single genotype of M avium subsp avium. The protocol included drugs advocated for the treatment of M avium infections in humans and cage birds. (2,13-15,18,21) This study mimicked the situation that most avian veterinarians would confront in attempting to treat 1 or more birds with mycobacteriosis without knowing the organisms' sensitivity to the antibiotics that are recommended for treatment.

Overall, this treatment protocol was considered to be ineffective. Nine of the I 1 doves that completed the 6 months of therapy were still infected and had significant lesions associated with their persisting infections. Furthermore, 5 of 16 birds considered infected died as the result of their mycobacterial infections before finishing therapy.

There are many potential explanations for treatment failure. In humans, treatment failure is usually the result of poor patient compliance, the presence of drug resistance, or both. (36) Poor compliance could not have been a contributing factor in this study because freshly prepared medications were administered to all of the birds every day at the same time by gavage. Antibiotic resistance, however, may have been a critical factor. Most isolates of M avium from humans have natural and acquired resistance to many antimycobacterial drugs. (37) Development of resistance to antibiotics used for the treatment of M intracellulare infection in little blue penguins (Eudyptula minor) has been also recently reported. (9) However, the natural or acquired resistance of M avium subsp avium isolates from birds to antimycobacterial drugs and the likelihood that it will acquire resistance to them during therapy has not been investigated in birds with mycobacteriosis. In our study, the 2 isolates cultured from these doves showed resistance to 2 of the 3 drugs used in this protocol. These findings highlight the importance of attempting to isolate and determine the antibiotic sensitivity of the Mycobacterium species that is being treated. In practice, as was the case in this study, many samples submitted for culture that are known to contain mycobacteria often prove culture negative and the turnaround time between when the samples are taken and the organism isolated or antibiotic resistance tested can be several months, (33) hampering its timely clinical application. Furthermore, the predictive value of in vitro evaluation of antibiotic resistance testing for M avium complex and other nontuberculous mycobacteria is controversial. (18,38) However, if an increased emphasis is made to determine resistance patterns in organisms identified, in the laboratory as well clinically, from birds with mycobacteriosis, this epidemiologic information would be invaluable for future therapeutic trails. Unfortunately, because of the small number of isolates, it was not possible to draw any conclusion from this study as to the development of antimicrobial resistance during treatment.

Other potential causes of treatment failure in these doves could include a failure of the drugs to reach therapeutic concentrations, a dosage interval that was too long, inability of the drugs to reach the organisms, and an inadequate duration of therapy. It is not possible to speculate on the effect of ethambutol and rifampin in these birds because pharmacokinetic trials have not been done with these drugs, and the ability of the dose used to reach effective tissue concentrations in birds is not known. In contrast, it is highly likely that the dose of the azithromycin used in this study resulted in effective tissue concentrations, at least in the relatively normal tissues. In mammals, azithromycin has been shown to achieve high tissue concentrations in the liver, spleen, and lungs that exceed those found in the blood. (39) Also, a dose of 40 mg/kg given every 48 hours was found to maintain therapeutic blood concentrations in blue and gold macaws (Ara ararauna) and to be effective at eliminating Chlamydophila psittaci infections in cockatiels (Nymphicus hollandicus). (40) Drug metabolism could vary among different avian species, and the lack of pharmacokinetic studies for these antimycobacterial drugs in doves precludes further conclusions about the ability of these drugs to reach therapeutic concentrations.

The lesions found in the doves at the end of this treatment trial were different from the lesions found in doves from the same flock that were not treated. (28) In the untreated birds, it was common to find organisms in macrophages and multinucleated giant cells, (28) whereas in this study, intracellular organisms were not seen. However, large numbers of organisms were still present in the necrotic center of granulomas. Antituberculous drugs may vary in their ability to kill mycobacteria, depending on whether they are located in cavitary or closed caseous lesions or within the cells. (41) These findings suggest that this combination of drugs used may have killed the intracellular mycobacteria but either did not achieve therapeutic concentrations, as previously discussed, or were not effective in this environment of the caseous cores of the granulomas.

In addition to the absence of mycobacteria in phagocytic cells of treated birds, lesions in the treated birds were less severe than those in untreated birds from the same flock. (28) The number of tissues where acid-fast organisms could be identified also was less in treated birds and most birds outwardly appeared healthy at the end of treatment. These findings may suggest that treatment was benefitting the birds that survived the course of therapy. Therefore, had treatment continued for a full year, as advocated for tuberculosis in humans, (36) a more successful outcome may have been achieved.

Histologic examination of liver biopsy samples has been advocated as an effective means of monitoring the success of antimycobacterial treatment. (13-15,21) Results of this study and those of a previous one (29) do not support this claim. In fact, only 1 in 11 of the birds that were still infected at the end of the treatment trial had acid-fast organisms in their liver identified by liver biopsy.

The combination of azithromycin, ethambutol, and rifampin at the doses used in this study appeared to be well tolerated by the healthy doves. Weight loss, optic neuritis, and significant changes in the plasma concentrations of AST and CK or uric acid plasma concentration were not observed. A steady increase observed in aspartate aminotransferase may be consistent with increased microsomal hepatic activity reported for rifampin. (37) Rifampin is a semisynthetic bactericidal drug and, together with ethambutol and macrolides like clarithromycin or azithromycin, is a first choice for treatment of M avium infections in humans. (36) Its mechanism of action is through inhibition of the DNA-dependant RNA polymerase and preventing transcription of mRNA and protein synthesis. Hepatotoxicity is a common adverse effect under prolonged use of this drug, and microsomal cytochrome P450 enzyme induction occurs, affecting the metabolism of different drugs. Monitoring of liver function is recommended in humans receiving this medication. Plasma concentrations of AST together with bile acids are usually evaluated to determine hepatic damage and function, respectively, in birds. (42) However, a significant increase in AST activity alone was not observed in this study.

This is the first comprehensive prospective clinical trial on the efficacy of a treatment protocol for avian mycobacteriosis caused by M avium subsp avium in a large group of birds. The ineffectiveness of this treatment protocol, as demonstrated by the presence of mycobacteria at least in 9 of the 11 birds and death of 5 of the infected birds before completion of treatment, indicates that eradication of the infection status is not easy to achieve. Furthermore, this study demonstrated that liver biopsy alone is not useful to evaluate treatment efficacy.

Our study shows a need for more information on drug resistance in the Mycobacterium species that infect birds and on the pharmacology in birds of the drugs that are typically used to treat them. Currently, there is not enough evidence-based and scientific knowledge to treat the avian patient effectively with antimycobacterial drugs. (43) We do not recommend at this time the medical treatment of birds affected with mycobacteriosis in uncontrolled clinical settings. Recommendations about the necessary research steps to effectively undertake the challenges associated with the treatment of mycobacterial infections in birds have been proposed. (43)

Acknowledgments'. Funding for this project was provided by the Schubot Exotic Bird Health Center, Texas A&M University, the Association of Avian Veterinarians, and The Smokey Mountain Bird Club. We are very grateful to Mrs Debra Turner for her assistance with this study. This manuscript was improved by comments and suggestions made by Drs Gagandeep Kaur and Hrvoje Smodlaka and Mr John Greenwood.

References

(1.) Hoop RK, Bottger EC, Pfyffer GE. Etiological agents of mycobacteriosis in pet birds between 1986 and 1995. J Clin Microbiol. 1996;34(4):991-992.

(2.) Katoch VM. Infections due to non-tuberculous mycobacteria (NTM). Indian J Med Res. 2004;120(4):290-304.

(3.) Steinmetz HW, Rutz C, Hoop RK, et al. Possible human-avian transmission of Mycobacterium tuberculosis in a green-winged macaw (Ara chloroptera). Avian Dis. 2006;50(4):641 645.

(4.) Bertelsen MF, Grondahl C, Giese SB. Disseminated Mycobacterium celatum infection in a white-tailed trogon (Trogon viridis). Avian Pathol. 2006;35(4):316-319.

(5.) Travis EK, Junge RE, Terrell SP. Infection with Mycobacterium simiae complex in four captive Micronesian kingfishers. J Am Vet Med Assoc. 2007;230(10): 1524-1529.

(6.) Schrenzel M, Nicolas M, Witte C, et al. Molecular epidemiology of Mycobacterium avium subsp. avium and Mycobacterium intracellulare in captive birds. Vet Microbiol. 2008; 126(1-3): 122-131.

(7.) Schmidt V, Schneider S, Schlomer J, et al. Transmission of tuberculosis between men and pet birds: a case report. Avian Pathol. 2008;37(6):589-592.

(8.) Manarolla G, Liandris E, Pisoni G, et al. Avian mycobacteriosis in companion birds: 20-year survey. Vet Microbiol. 2009; 133(4):323-327.

(9.) Napier JE, Hinrichs SH, Lampen F, et al. An outbreak of avian mycobacteriosis caused by Mycobacterium intracellulare in little blue penguins (Eudyptula minor). J Zoo Wildl Med. 2009;40(4):680-686.

(10.) Schrenzel MD. Molecular epidemiology of mycobacteriosis in wildlife and pet animals. Vet Clin North Am Exot Anim Pract. 2012; 15(1): 1-23.

(11.) Dorrestein GM. Bacteriology. In: Altman RB, Clubb SL, Dorrestein GM, Quesenberry K, eds. Avian Medicine and Surgery. Philadelphia, PA: WB Saunders; 1997:255-280.

(12.) Phalen DN. Avian mycobacteriosis. In: Bonagura JD, ed. Kirk's Current Veterinary Therapy XIII. Philadelphia, PA: WB Saunders; 2000:1116-1118.

(13.) Pollock CG. Implications of mycobacteria in clinical disorders. In: Harrison GJ, Lightfoot TL, eds. Clinical Avian Medicine. Vol 2. Palm Beach, FL: Spix Publishing; 2006:681-688.

(14.) Lennox AM. Successful treatment of mycobacterial infection in three psittacine birds. Proc Annu Conf Assoc Avian Vet. 2002:111-114.

(15.) Lennox AM. Mycobacteriosis in companion psittacine birds: a review. J Avian Med Surg. 2007;21(3): 181-187.

(16.) Gerlach H. Bacteria. In: Ritchie BW, Harrison GJ, Harrison LR, eds. Avian Medicine: Principles and Application. Lake Worth, FL: Wingers Publishing; 1997:949-983.

(17.) Tell LA, Woods L, Cromie RL. Mycobacteriosis in birds. Rev Sci Tech. 2001 ;20(1): 180-203.

(18.) Griffith DE. Therapy of non-tuberculous mycobacterial disease. Curr Opin Infect Dis. 2007;20(2): 198-- 203.

(19.) Samour J. Avian Medicine. New York, NY: Mosby, Elsevier; 2007.

(20.) Esteban J. Ortiz-Perez A. Current treatment of atypical mycobacteriosis. Expert Opin Pharmacother. 2009;10(17):2787-2799.

(21.) Van der Heyden N. Clinical manifestations of mycobacteriosis in pet birds. Sem Avian Exot Pet Med. 1997;6(1): 18-24.

(22.) Beynon PH, Forbes NA, Harcourt-Brown NH. BSA VA Manual of Raptors, Pigeons and Waterfowl. BSAVA Publishing: Cheltenham, United Kingdom; 1996.

(23.) Hershfield E. Tuberculosis: treatment. Can Med Assoc J. 1999; 161 (4):405-411.

(24.) Biet F, Boschiroli ML, Thorel MF, Guilloteau LA. Zoonotic aspects of Mycobacterium bovis and Mycobacterium avium-intracellulare complex (MAC). Vet Res. 2005;36(3):411-436.

(25.) Iwamoto T, Nakajima C, Nishiuchi Y, et al. Genetic diversity of Mycobacterium avium subsp. hominissuis strains isolated from humans, pigs, and human living environment. Infect Genet Evol. 2012; 12(4):846-852.

(26.) Jun HJ, Lee NY, Kim J, Koh WJ. Successful treatment of Mycobacterium celatum pulmonary disease in an immunocompetent patient using antimycobacterial chemotherapy and combined pulmonary resection. Yonsei Med J. 2010;51 (6):980 983.

(27.) Foldenauer U, Curd S, Zulauf I, Hatt JM. Antemortem diagnosis of mycobacterial infection by liver biopsy in a budgerigar (Melopsittacus undulatus). Schweiz Arch Tierheilkd. 2007;149(6):273276.

(28.) Saggese MD, Tizard I, Phalen DN. Mycobacteriosis in naturally infected ring-neck doves (Streptopelia risoria): investigation of the association between feather colour and susceptibility to infection, disease and lesions type. Avian Pathol. 2008;37(4):443-150.

(29.) Saggese MD, Tizard I, Phalen DN. Comparison of sampling methods, culture, acid-fast stain, and polymerase chain reaction assay for the diagnosis of mycobacteriosis in ring-neck doves (Streptopelia risoria). J Avian Med Surg. 2010;24(4):263-271.

(30.) Gray PL, Saggese MD, Phalen DN, Tizard I. Humoral response to Mycobacterium avium subsp. avium in naturally infected ring-neck doves (Streptopelia risoria). Vet Immunol Immunopathol. 2008; 125(3-4):216-224.

(31.) Dahlhausen B, Soler-Tovar D, Saggese MD. Diagnosis of mycobacterial infections in the exotic pet patient with emphasis on birds. Vet Clin North Am Exot Anim Pract. 2012;15(1):71-83.

(32.) Friedman LM, Furberg CD, DeMets DL. Fundamentals of Clinical Trials. 3rd ed. New York, NY: Springer; 1998.

(33.) Mahon CR, Manuselis G Jr, Lehman DC. Textbook of Diagnostic Microbiology. 3rd ed. New York, NY: Elsevier; 2006.

(34.) Morita Y, Maruyama S, Kabeya H, et al. Genetic diversity of the dnaJ gene in the Mycobacterium avium complex. J Med Microbiol. 2004;53(Pt 8):813-817.

(35.) Thompson JD, Gibson TJ, Plewniak F, et al. The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 1997;25(24):4876-4882.

(36.) Cole ST, Eisenach KD, McMurray DN, Jacobs WR Jr. Tuberculosis and the Tubercle Bacillus. Washington, DC: ASM Press; 2004.

(37.) Tomioka H. Present status and future prospects of chemotherapeutics for intractable infections due to Mycobacterium avium complex. Curr Drug Discov Technol. 2004;1(4):255 268.

(38.) van Ingen J, Boeree MJ, van Soolingen D, Mouton JW. Resistance mechanisms and drug susceptibility testing of nontuberculous mycobacteria. Drug Resist Updat. 2012; 15(3): 149-161.

(39.) Yoshida H, Furuta T. Tissue penetration properties of macrolide antibiotics--comparative tissue distribution of erythromycin-stearate, clarithromycin, roxithromycin and azithromycin in rats. Jpn J Antibiot.1999-52(7):491-503.

(40.) Carpenter JW, Olsen JH, Randle-Port M, et al. Pharmacokinetics of azithromycin in the blue and gold macaw (Ara ararauna) after intravenous and oral administration. J Zoo Wildl Med. 2005;36(4):606-609.

(41.) Scholar EM, Pratt WB. The Antimicrobial Drugs. New York, NY: Oxford University Press; 2000.

(42.) Fudge AM. Laboratory Medicine: Avian and Exotic Pets. New York, NY: WB Saunders; 2000.

(43.) Buur J, Saggese MD. Taking a rational approach in the treatment of avian mycobacteriosis. Vet Clin North Am Exot Anim Pract. 2012; 15(1):57-70.

Miguel D. Saggese, DVM, MS, PhD, Ian Tizard, BVMS, PhD, Patricia Gray, DVM, MS, and David N. Phalen, DVM, PhD, Dipl ABVP (Avian)

From The Schubot Exotic Bird Health Center, Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, 4467 TAMU, College Station, TX 77843-4467, USA. Present address: College of Veterinary Medicine, Western University of Health Sciences, 309 E Second St, Pomona, CA 91766-1854, USA (Saggese); and The Wildlife Health and Conservation Centre, University of Sydney, Camden, New South Wales, Australia, 4475 (Phalen).

Table 1. Comparison of sensitivity (% test positive) of different
organ sampling and techniques used for the diagnosis of
mycobacteriosis in 11 ring-neck doves with spontaneous mycobacteriosis
treated with azithromycin, ethambutol, and rifampin for 180 days.
Liver, spleen, and lung samples were collected at postmortem
examination immediately after euthanasia. Additionally, biopsy samples
of liver and bone marrow from the ulna were collected immediately
after euthanasia by the same techniques as used in a live bird. All
percentages are rounded.

Technique       Liver, %   Spleen, %   Lung, %   Bone marrow
                                                  biopsy, %

Ziehl-Neelsen    9           45.5      27.3           0
Culture         18.2         45.5      0              9
PCR              9.1         27.3      -- (a)         0
Combined        27.3         61.5      27.3           9

Technique         Liver     Combined, %
                biopsy, %

Ziehl-Neelsen    9             90.1
Culture         18.2           54.5
PCR              0             36.4
Combined        27.3

(a) PCR was not performed on lung samples.
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Author:Saggese, Miguel D.; Tizard, Ian; Gray, Patricia; Phalen, David N.
Publication:Journal of Avian Medicine and Surgery
Article Type:Clinical report
Geographic Code:1USA
Date:Dec 1, 2014
Words:6447
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