Journal of Zoo and Wildlife Medicine 37(4): 498–512, 2006 Copyright 2006 by American Association of Zoo Veterinarians

MYCOBACTERIUM AVIUM IN PYGMY (BRACHYLAGUS IDAHOENSIS): 28 CASES

Lisa A. Harrenstien, D.V.M., Dipl. A.C.Z.M., Mitchell V. Finnegan, D.V.M., Nina L. Woodford, D.V.M., M.P.H., Dipl. A.C.L.A.M., Kristin G. Mansfield, D.V.M., M.P.V.M., W. Ray Waters, D.V.M., Ph.D., John P. Bannantine, Ph.D., Michael L. Paustian, Ph.D., Michael M. Garner, D.V.M., Dipl. A.C.V.P., Antony C. Bakke, Ph.D., Charles A. Peloquin, Pharm.D., and Terry M. Phillips, Ph.D., D.Sc.

Abstract: The Columbia basin subpopulation of pygmy Brachylagus idahoensis was listed as endangered by the United States Fish and Wildlife Service in November 2001, and no pygmy rabbits have been seen in the wild since spring 2002. Captive propagation efforts have attempted to increase population size in preparation for reintroduction of into central Washington. Disseminated mycobacteriosis due to Mycobacterium avium has been the most common cause of death of adult captive pygmy rabbits. Between June 2002 and September 2004, mycobacteriosis was diagnosed in 28 captive adult pygmy rabbits (representing 29% of the captive population), in contrast to 18 adult pygmy rabbits dying of all other causes in the same time period. Antemortem and postmortem medical records were evaluated retrospectively to describe the clinical course of mycobacteriosis in pygmy rabbits, physical examination findings, and diagnostic test results in the diagnosis of mycobacteriosis in pygmy rabbits. Various treatment protocols, possible risk factors for mortality, and recommendations for prevention of mycobacteriosis were evaluated also. Com- promised cell-mediated immunity appears to be the best explanation at this time for the observed high morbidity and mortality from mycobacterial infections in pygmy rabbits. Key words: Brachylagus idahoensis, cell-mediated immunity, mycobacteriosis, Mycobacterium avium complex, .

INTRODUCTION fined to the basin of the Columbia River, is esti- The pygmy rabbit (Brachylagus idahoensis)is mated to have been isolated geographically from the only species in the genus Brachylagus and has other populations of the species for at least 10,000 been found in isolated populations in southeastern yr and exhibits less genetic diversity than do other Washington, southern Idaho, southwestern Mon- populations.21,22,42,43 Adult pygmy rabbits weigh ap- tana, western Wyoming, western Utah, northern proximately 400 g and display several dietary and Nevada, northeastern , and eastern behavioral traits that distinguish them from cotton- .9,14,26 The population in Washington, con- tails (Sylvilagus spp.), jackrabbits (Lepus spp.), and Old World rabbits (Oryctolagus sp.). From the Oregon Zoo, 4001 Southwest Canyon Road, In the last decade, pygmy rabbit populations Portland, Oregon 97221, USA (Harrenstien, Finnegan); have declined in Washington and other states where Office of the Campus Veterinarian, Washington State Uni- sagebrush has been burned, has been con- versity College of Veterinary Medicine, Pullman, Wash- verted to agriculture, or has been cleared from large ington 99164, USA (Woodford); Washington Department areas and replaced with bunch grasses that increase of Fish and Wildlife, 2315 North Discovery Place, Spo- livestock forage opportunities. The wild Columbia kane Valley, Washington 99216, USA (Mansfield); Bac- Basin pygmy rabbit (CBPR) population declined terial Diseases of Livestock Research Unit, National An- precipitously from about 150 rabbits in 1995 to imal Disease Center, Agricultural Research Service, Unit- fewer than 30 in 2001.16,42 Fearing extinction of the ed States Department of Agriculture, 2300 Dayton Ave- nue, Ames, Iowa 50010, USA (Waters, Bannantine, CBPR, the Washington Department of Fish and Paustian); Northwest ZooPath, 654 West Main Street, Wildlife initiated a captive breeding program in co- Monroe, Washington 98272, USA (Garner); Department operation with the Washington State University of Pathology and Medicine, Oregon Health and Science College of Veterinary Medicine (WSU), Oregon University, Portland, Oregon 97239, USA (Bakke); De- Zoo (OZ), and Northwest Trek Zoological Park partment of Medicine, National Jewish Medical and Re- (NWT). In December 2000, four wild pygmy rab- search Center, and University of Colorado Schools of bits from a nonendangered population in the Lemhi Pharmacy and Medicine, Denver, Colorado 80206, USA (Peloquin); and the National Institutes of Health, 9000 Valley in Idaho were brought to OZ for the purpose Rockville Pike, Bethesda, Maryland 20892, USA (Phil- of developing pygmy rabbit husbandry protocols. lips). Correspondence should be directed to Dr. Harren- Sixteen endangered CBPR were captured from stien. 2001–2002 to serve as founders for captive breed- 498 HARRENSTIEN ET AL.—MYCOBACTERIUM AVIUM IN PYGMY RABBITS 499 ing, and in 2002, both WSU and OZ began breed- MATERIALS AND METHODS ing CBPR for eventual reintroduction. The United Clinical cases States Fish and Wildlife Service listed the CBPR under emergency provisions of the Endangered Twenty-eight cases of fatal disseminated myco- Species Act on November 30, 2001,39 with full list- bacteriosis due to M. avium (29% of the captive ing status on March 5, 2003.40 Presently, there are adult population) were diagnosed in captive pygmy 19 CBPR, 12 Idaho pygmy rabbits, and 24 inter- rabbits between June 2002 and September 2004 at cross pygmy rabbits of CBPR–Idaho origin in cap- two of the three captive holding facilities. Staff at tive breeding programs in Washington and Oregon. OZ and/or WSU managed clinical cases. A variety No CBPR have been observed in wild habitat since of local and reference laboratories performed di- spring 2002. agnostic tests. Antemortem and postmortem medi- ϭ Disseminated infection with Mycobacterium av- cal records from all cases (n 28) were evaluated ium complex has been the most common cause of retrospectively by one author (LH). Postmortem di- death in adult captive pygmy rabbits, affecting agnoses from all other captive adult pygmy rabbit mortalities at all captive holding facilities during those of all origins. In most mammalian species, the same time period (n ϭ 18) were summarized including rabbits, M. avium is not pathogenic un- for comparison. less the immune system of the patient is compro- mised. Domestic rabbits (Oryctolagus cuniculus) Mycobacterial cultures have rarely been reported to have naturally-ac- Antemortem mycobacterial surveillance of the quired mycobacteriosis, and such cases usually OZ captive pygmy rabbit population was conducted have been associated with heavily-contaminated for 13 mo using bimonthly fecal mycobacterial cul- pastures upon which cattle infected with M. bovis tures from each rabbit. Mycobacterial culture was or M. avium paratuberculosis had been living be- performed using standard microbiological tech- fore the rabbits arrived.13,17 General causes for im- niques. Molecular diagnostic testing such as poly- munosuppression may include psychological stress, merase chain reaction (PCR) amplification or se- inbreeding, viral infection, and drug therapy. De- quencing of DNA of isolates was performed several pressed cell-mediated immune function has been weeks into the culture period for final placement of reported as the cause for increased morbidity and the mycobacterial isolate into one of three catego- mortality of M. avium infections in marsupials, hu- ries (M. tuberculosis complex, M. avium complex, mans infected with HIV, and organ transplant pa- or ‘‘other’’). tients.5,7,15,18,23,25,27,34 In humans with AIDS, mycobacteria may be ac- Gross and histopathologic examinations quired via gastrointestinal and respiratory routes Histopathologic examination was necessary for through contaminated airborne (coughed or confirmation of diagnosis of disseminated myco- sneezed) particles, feces, urine, genital discharges, bacteriosis. Biopsy and necropsy tissues were col- 24,41 milk, feed, or water. Sometimes leading to in- lected by several prosectors and were preserved in fection, M. avium may colonize or may pass 10% neutral buffered formalin. Tissues were pro- through the gastrointestinal tract of avian species cessed routinely, sectioned at 5 ␮m, mounted on and may be transmitted intermittently in feces.20,38 glass microscope slides, and stained with hematox- This retrospective study was initiated due to an ylin and eosin. Mycobacteriosis-associated lesions apparent high incidence of mycobacteriosis in the were typically histiocystic, granulomatous, and/or captive pygmy rabbit population. Antemortem and necrotizing. On average, two lesions per case were postmortem medical records from 28 cases of my- stained with Fite’s acid-fast technique to confirm cobacteriosis were evaluated retrospectively in or- presence of acid-fast bacilli. der to describe the clinical course of the disease in pygmy rabbits, to determine the relative value of Drug choices physical examination and diagnostic tests in the di- Clinicians typically made initial drug choices be- agnosis of mycobacteriosis in pygmy rabbits, to de- fore a diagnosis of mycobacteriosis had been con- scribe the use of various treatment protocols, and firmed. Several antibiotics, often in combination, to assess possible risk factors for the observed high were used for treatment in 16 of the 28 pygmy rab- mortality. Development of husbandry and medical bits during the time period of their clinical signs. management recommendations for prevention of Azithromycin was administered to 12 rabbits, at 50 mycobacteriosis in pygmy rabbits was a goal of this mg/kg p.o. q 24 hr (Zithromax 40 mg/ml oral sus- retrospective study. pension, Pfizer Labs, New York, New York 10017, 500 JOURNAL OF ZOO AND WILDLIFE MEDICINE

USA). Rifampin was administered to nine rabbits, for determination of important antigenic proteins at 40 mg/kg p.o. q 12 hr (160 mg/ml compounded that could be used in future mycobacterial surveil- oral suspension, University Compounders Inc., lance. Immunoblot assays were performed accord- Portland, Oregon 97203, USA). Enrofloxacin was ing to previously reported procedures, using a administered to six rabbits, at 15 mg/kg p.o. q 24 whole cell sonicate of a pygmy rabbit M. avium hr or 5–21 mg/kg s.c. q 24 hr (Baytril 22.7 mg/ml strain,1,44 as well as serum and plasma samples from injectable solution, Bayer Healthcare, Shawnee pygmy rabbits against the whole cell sonicate. Se- Mission, Kansas 66201, USA). Ethambutol was ad- rum and plasma samples included 30 samples from ministered to four rabbits, at 45 mg/kg p.o. q 12 hr 26 rabbits presumed to be healthy and 23 samples (200 mg/ml compounded oral suspension, Univer- from 11 animals currently infected with mycobac- sity Compounders). Rifabutin was administered to teria. 3 rabbits at 25 mg/kg p.o. q 12 hr (40 mg/ml com- pounded oral suspension, University Compound- Assessment of cell-mediated immunity and ers). Trimethoprim-sulfadiazine was administered cytokines to two rabbits at 30 mg/kg i.m. q 12–24 hr (480 The cell-mediated immunocompetence of pygmy mg/ml compounded injectable suspension, Univer- rabbits was evaluated. Heparinized blood samples sity Compounders). Dosages for these drugs were were collected from 51 captive pygmy rabbits (ages extrapolated from published references for domes- 3–50 mo), as well as from domestic laboratory rab- 2,6,11,36 tic rabbits. bits (Oryctolagus cuniculus, n ϭ 5, adult), and from Although drug susceptibility testing was not pre- a California riparian brush rabbit (Sylvilagus bach- dicted to be clinically relevant for individual case mani riparius, n ϭ 1, adult). Lymphocytes and management due to the length of time required for plasma from each sample were used for cell-me- completion of testing (2–3 mo), seven postmortem diated immunity assays (lymphocyte transformation M. avium isolates from pygmy rabbit necropsies with mitogens, cell–cell interaction of mixed lym- were assessed for in vitro minimum inhibitory con- phocyte cultures), and cytokine measurement. Lym- centration susceptibility to various medications, in- phocyte transformation studies were performed as cluding amikacin, azithromycin, ciprofloxacin, cla- previously described for the Matschie’s tree kan- rithromycin, ethambutol, ethionamide, rifabutin, ri- garoo (Dendrolagus matschiei).25 Mixed lympho- fampin, and streptomycin. cyte cultures to study cell–cell interaction were per- Drug concentration measurement formed in a similar manner, using allogeneic ani- Banked sera and/or plasma samples from three mals instead of plant mitogens as stimulators. Cy- clinical cases were assayed for azithromycin, rifam- tokines were measured in rabbit plasma by pin, rifabutin, and ethambutol concentrations to de- immunoaffinity capillary electrophoresis using iso- termine whether therapeutic concentrations of these lated rabbit cytokine standards in a similar manner antimicrobials could be achieved in pygmy rabbits. to that previously described for human cyto- 32,33 Samples were collected 2–3 hr after oral adminis- kines. Numerical data were interpreted to be sig- tration of the drug, were stored frozen at Ϫ80ЊC, nificantly different if mean values were associated Ͻ and were shipped frozen overnight to National Jew- with P 0.01, using Student’s t-test. ish Medical and Research Center (Denver, Colora- RESULTS do 80206, USA) for analysis. All assays were per- formed using validated high-performance liquid Clinical cases chromatography (azithromycin, rifampin, rifabutin) or gas chromatography/mass spectrometry (etham- Mycobacteriosis was diagnosed definitively an- ϭ ϭ butol) methods that met the criteria of the College temortem (n 9) or postmortem (n 19) in pygmy of American Pathologists. Standard curves had the rabbits with histologic lesions that were positive for following ranges: azithromycin, 0.05–5 ␮g/ml; ri- acid-fast bacilli and/or histologic lesions from fampin, 0.50–50 ␮g/ml; rifabutin, 0.10–2 ␮g/ml; which mycobacteria were cultured. and ethambutol, 0.20–10 ␮g/ml.30,31 Six-point stan- The gender distribution of mycobacteriosis cases dard curves and at least three quality control sam- was biased slightly toward females (71%, 20 fe- ples were assayed with the research samples. males of 28 total mycobacteriosis cases), compared with the gender distribution of nonmycobacteriosis Assessment of mycobacterial antibody captive adult mortalities (56%, 10 females of 18 production total nonmycobacteriosis mortalities). Mycobacte- Mycobacterial antibodies were evaluated retro- riosis was seen in both wild-caught (n ϭ 10) and spectively in healthy and infected pygmy rabbits captive-born (n ϭ 18) animals. The wild-caught an- HARRENSTIEN ET AL.—MYCOBACTERIUM AVIUM IN PYGMY RABBITS 501 imals that died of mycobacteriosis were in captivity of the rabbits tested (39% of the samples), with for 12–30 mo before death. relatively equal numbers of anemic rabbits showing The average age at time of onset of clinical signs, normocytic normochromic nonregenerative versus when present, due to mycobacteriosis was 28.5 mo, macrocytic hypochromic regenerative red cell in- with the earliest onset of clinical signs observed in dices. Six rabbits had nucleated red blood cells in a 10.5-mo-old intercross rabbit. Healthy pygmy their peripheral blood smears, despite a normal or rabbits have been known to live longer than 4 yr increased hematocrit, and one had metamyelocytes in captivity, regardless of population origin (L. Har- in a peripheral blood smear, despite lack of noted renstien, unpubl. data). postmortem histologic lesions in bone marrow. Ap- The nonspecific clinical signs of weight loss, proximately one half of the biochemistry panels re- lethargy, perineal soiling, and anorexia were those vealed hypoalbuminemia and/or hyperglobulinemia most commonly reported (64%, 50%, 39%, and (decreased albumin/globulin ratio). Hypercholester- 32% of cases, respectively), with other signs being olemia and azotemia were noted less frequently. El- evident in less than 20% of cases. Lameness due to evation of alkaline phosphatase concentration, osteomyelitis was seen in 14% of the rabbits in this which sometimes occurs in cases of osteolysis, was study. Many clinical signs could be associated with only noted in one with osteomyelitis. pathophysiology typical of mycobacteriosis in other Mycobacteriosis mortalities occurred at OZ and species (e.g., pale mucous membranes associated WSU. Adult mortalities at the OZ were typically with anemia of chronic inflammation, dyspnea as- due to mycobacteriosis (72%, 13 of 18 total adult sociated with pneumonia). However a few were un- mortalities) versus other etiologies; adult mortali- expected, including lactation in three nonpregnant ties at WSU were less frequently due to mycobac- rabbits (11% of cases). teriosis (54%, 15 of 28 total adult mortalities). One Ultrasonographic examination of liver and kid- mortality due to mycobacteriosis occurred at WSU ney parenchyma was not useful for detection of he- 2 wk after the animal had been transferred from patic and renal granulomas. Radiographic findings NWT, and four rabbits strongly suspected to have of pneumonia, osteomyelitis, or soft tissue masses mycobacteriosis were transferred from WSU to OZ, did not change during the course of disease in three where they were euthanized. animals that underwent five sets of radiographs A greater percentage of mortalities were attri- over a 2-mo period. If abnormal lung, bone, or a buted to mycobacteriosis in captive CBPR than in soft tissue mass was localized during physical or captive Idaho rabbits, despite a similar age range radiographic examination, a biopsy was performed of these populations (Table 2). and the collected material was cultured or stained for acid-fast organisms. Only 32% of eventually Mycobacterial cultures confirmed mycobacteriosis cases (9 of 28 cases, Ta- Eleven OZ mycobacteriosis mortalities had an- ble 1) showed direct evidence of mycobacterial in- temortem fecal mycobacterial cultures performed; fection before death, either as acid-fast positive ba- only one animal was fecal positive, despite six of cilli in a microscopic exam of a lesion aspirate or the 11 animals showing intestinal granulomas at as positive mycobacterial culture of blood (3 of 4 necropsy. Three animals previously culture-nega- animals tested), urine (7 of 8 animals tested), feces tive generated culture-positive urine or blood sam- (3 of 15 animals tested), or an abnormal mass (4 ples after 2–10 wk of antibiotic therapy. of 9 animals tested). Given that mycobacterial cul- tures require several weeks to complete, some of Gross and histopathologic examinations these antemortem positive findings were not known Gross lesions due to mycobacteriosis were typi- until after death. cally yellow, moderately firm (slightly firmer than Diagnostic blood analyses (complete blood count normal fat), and of variable size up to several mil- [CBC] and plasma or serum biochemistry panel) limeters. The number and size of lesions seen dur- were performed for 57% of mycobacteriosis rabbits ing gross necropsy did not correlate with the degree at some point during their illness, and their data of clinical debilitation. For example, 27 rabbits ex- were compared to reference ranges generated from hibited pulmonary lesions histologically, although presumed healthy pygmy rabbits at OZ (Table 1). just 18 rabbits showed pulmonary lesions in gross Ninety-one percent of mycobacteriosis rabbits postmortem examination and few rabbits had been showed abnormal CBC values at some point during diagnosed antemortem with pulmonary disease. illness, with heterophilia being most common. Mycobacterial lesions were found histologically in Monocytosis was seen in 44% of the rabbits tested liver and kidney in more than 50% of the cases. (29% of the samples). Anemia was present in 38% Overall, 19 tissues were found histologically to 502 JOURNAL OF ZOO AND WILDLIFE MEDICINE

Table 1. Antemortem diagnostic procedures and results reported in medical records of 28 adult pygmy rabbits (Brachylagus idahoensis) that died of disseminated mycobacteriosis originating from Columbia Basin, Idaho, or Co- lumbia Basin–Idaho intercross populations, 2001–2004.

Rabbits Rabbits testedb positivec Test positived Diagnostic test n (%) n (%) n (%)

Radiography 15 (54) 10 (67) 21 of 33 (64) Osteolysis 6 (40) 14 of 33 (42) Pneumonia 4 (27) 4 of 33 (12) Aspirate (lesion) cytology 9 (32) 4 (44) 4 of 9 (44) Transtracheal lavage acid-fast cytology 1 (4) 0 (0) 0 (0) Ultrasonography (hepatic/renal) 6 (21) 0 (0) 0 (0) Complete blood counta 16 (57) Leukocytosis (Ͼ11,443/␮l) 11 (69) 16 of 35 (46) Absolute heterophilia (Ͼ5,653/␮l) 12 (75) 24 of 35 (69) Absolute monocytosis (Ͼ505/␮l) 7 (44) 10 of 35 (29) Absolute lymphocytosis (Ͼ5,164/␮l) 4 (25) 4 of 35 (11) Absolute lymphopenia (Ͻ1,332/␮l) 3 (19) 5 of 35 (14) Thrombocytopenia (qualitative) 2 (13) 2of34(6) Anemia (hematocrit Ͻ37%) 6 (38) 13 of 33 (39) Նone hematologic abnormality 14 (88) 32 of 35 (91) Serum/plasma biochemistry panela 16 (57) Low albumin/globulin ratio (Ͻ1.2) 16 (57) 12 (75) 17 of 33 (52) Hypercholesterolemia (Ͼ45 mg/dl) 13 (46) 10 (77) 11 of 18 (61) Elevated alkaline phosphatase (Ͼ146 IU/l) 16 (57) 1 (6) 1of33(3) Azotemia (elevated blood urea nitrogen Ͼ38 mg/dl) 16 (57) 3 (19) 6 of 34 (18) Elevated creatinine (Ͼ1.0 mg/dl) 16 (57) 2 (13) 4 of 34 (12) Ն one biochemical abnormality 15 (94) 24 of 34 (71) Fecal mycobacterial culturee 15 (54) 3 (20) 3 of 22 (14) Urine mycobacterial culturee 8 (29) 7 (88) 10 of 16 (63) Blood mycobacterial culturee 4 (14) 3 (75) 3 of 10 (30) Acid-fast bacilli found, or positive mycobacterial culture, in any antemortem sample 9 (32) Immunoblot of plasma/serum for Mycobacterium avium 7 (25) 5 (71) 16 of 19 (84) antibodye ‘‘positive,’’ ‘‘positive,’’ 2 (29) 3 of 19 (16) ‘‘suspect’’ ‘‘suspect’’

a Hematologic and biochemical reference ranges were established as the mean Ϯ 2 SD from healthy adult captive pygmy rabbits, and sample values were considered abnormal if Ն2 SD above or below the reference population’s mean value. b Rabbits tested: n ϭ number of rabbits that were evaluated by the listed diagnostic test, % ϭ percentage of rabbits evaluated compared with 28 rabbits with disseminated mycobacteriosis. c Rabbits positive: n ϭ number of rabbits in which an abnormal result ever was found; % ϭ percentage of rabbits positive com- pared with the number of rabbits ever receiving that test. d Test positive: n ϭ number of tests in which an abnormal result was found compared with the total number of tests performed; % ϭ percentage of abnormal test results compared with the number of tests performed. e Յ6 mo before illness was first noticed. contain mycobacterial lesions in the rabbits of this ate giant cells were present at the margin of ne- study. crotic tissue and inflammation in the granulomas. Histologically, mycobacterial lesions in pygmy Fite’s acid-fast stained sections revealed low to rabbits included granulomatous inflammation, dis- moderate numbers of acid-fast bacilli in the foci of crete granulomas, and areas of necrosis. Inflam- necrosis and sometimes within the cytoplasm of mation was predominantly comprised of macro- macrophages or multinucleate cells (Fig. 2). phages with fewer neutrophils, lymphocytes, and plasma cells, sometimes organized into discrete Drug choices granulomas oriented around foci of caseous necro- Drug susceptibility results showed similar, but sis (Fig. 1). Occasional Langhans-type multinucle- not identical, profiles. Most of the seven isolates HARRENSTIEN ET AL.—MYCOBACTERIUM AVIUM IN PYGMY RABBITS 503

Table 2. Causes of mortality of captive adult pygmy rabbits (Brachylagus idahoensis) originating from Columbia Basin (CBPR), Idaho, or Columbia Basin–Idaho intercross populations, 2001–2004, determined by postmortem gross and histopathologic evaluation.

Cause of pygmy CBPR Idaho Intercross All rabbit mortality na (%)b na (%)b na (%)b na (%)b

Mycobacteriosis 19 (70) 8 (47) 1 (50) 28 (61) Enterocolitis, nonparasitic 4 (15) 2 (12) 0 6 (13) Trauma 2 (7) 3 (18) 1 (50) 6 (13) Cysticercosis 0 1 (6) 0 1 (2) Nonmycobacterial abscess 0 1 (6) 0 1 (2) Neoplasia 1 (4) 0 0 1 (2) Capture complication 1 (4) 0 0 1 (2) Unknown 0 2 (12) 0 2 (4) Mortality totals by population 27 17 2 46

a n ϭ number of adult animals of the designated population in which the stated cause of mortality was found. b % ϭ percentage of the total number of necropsied adult animals of the designated population that died of that particular cause.

Figure 1. Brain, pygmy rabbit (Brachylagus idahoensis) with mycobacteriosis. Note granuloma in neuropil with core of necrosis (n) surrounded by macrophages and Langhans type multinucleate giant cells (arrows). Hematoxylin and eosin stain, bar ϭ 500 ␮m. 504 JOURNAL OF ZOO AND WILDLIFE MEDICINE

Figure 2. Brain, pygmy rabbit (Brachylagus idahoensis) with mycobacteriosis. Note large numbers of bacilli in the cytoplasm of a Langhans-type multinucleate giant cell. Fite’s acid fast stain, bar ϭ 60 ␮m.

tested were susceptible to azithromycin, clarithro- Assessment of mycobacterial antibody mycin, ethambutol, and rifabutin in vitro; therefore, production these drugs were preferentially used for treatment of later cases. Subjectively, antibiotic treatment ap- Fifty-three archived serum or plasma samples peared to improve or to stabilize the clinical status were evaluated by immunoblot to detect immuno- of an affected rabbit, with weight gain and cessa- globulin reactive to a M. avium whole cell sonicate. tion of lesion growth. In general, pygmy rabbits with active mycobacterial infection showed M. avium–specific antibody in Drug concentration measurement immunoblots, and noninfected pygmy rabbits were A preliminary analysis of achieved serum con- M. avium immunoblot negative. As illustrated in centrations was performed for azithromycin, rifam- Figure 3, analysis of serum obtained from a M. av- pin, rifabutin, and ethambutol. Azithromycin and ium infected rabbit showed reactivity to proteins of ϳ ϳ rifabutin serum or plasma concentrations ap- 100 kDa and 42 kDa in M. avium whole cell proached human target ranges (0.2–0.7 ␮g/ml and sonicate. Responses were not detected in serum ob- 0.3–0.9 ␮g/ml, respectively), with three rifabutin tained from a noninfected rabbit. Analysis of serial concentrations exceeding those typically sought in samples from another infected rabbit showed re- humans. Conversely, at the time points measured, activity to proteins of ϳ100 kDa, ϳ42 kDa, and rifampin and ethambutol serum or plasma concen- ϳ32–37 kDa in M. avium whole cell sonicate. Re- trations were well below the human target ranges sponses were not detected in serum obtained from (8–24 ␮g/ml and 2–6 ␮g/ml, respectively). this rabbit prior to infection, 2 yr earlier. HARRENSTIEN ET AL.—MYCOBACTERIUM AVIUM IN PYGMY RABBITS 505

tokines were reduced, whereas T helper 2 (Th2) cytokines were increased, indicating a Th2 polari- zation of the immune response.

DISCUSSION Captive pygmy rabbits appear to be extremely susceptible to infection from M. avium complex that progresses to fatal disease. The pygmy rabbits of our study acquired mycobacterial infections from unknown sources. We suspect substrate contami- nated with M. avium to be the source of infection, based on the ubiquitous natural prevalence of the organism. Soil substrate used in captive pygmy rab- bit enclosures is not sterilized before use; it is also possible that small birds could gain entry to captive enclosures, despite wire mesh walls and ceilings. Based on our retrospective analysis, it was not possible to determine with confidence whether rab- bits were colonized first in the wild or in captivity, Figure 3. Preparative immunoblot of Mycobacterium although the long interval between capture and on- avium pygmy rabbit isolate probed with three sequential set of disease (mean, 20 mo) suggests that infection blood samples from a pygmy rabbit (Brachylagus ida- occurred in captivity. hoensis) with clinical suspicion of mycobacteriosis. Lane 1: a pygmy rabbit diagnosed with M. avium infection; lane Comparison of disease prevalence between cap- 2: a pygmy rabbit known to be free of M. avium infection; tive facilities is difficult, because pygmy rabbits lane 3: clinical suspect, archive sample from 2 yr prior to were transferred frequently between facilities and onset of clinical signs of illness; lane 4: same clinical the course of mycobacteriosis is presumed to be suspect, at time of onset of clinical signs of illness; lane long. At each facility, occasional acid-fast granu- 5: same clinical suspect, 3 wk after onset of clinical signs lomas were noted on routine necropsy screening of of illness. Protein size markers are indicated in kilodaltons a few other captive animals of other species, how- in the left margin. ever, cultures of the tissues were not submitted so additional information regarding a presumed My- cobacterium species is not available. Animal keep- Assessment of cell-mediated immunity and ers, equipment, and exhibits in each instance were cytokines not associated with pygmy rabbit housing. In lymphocyte mitogen and antigen stimulation Although clinical signs of mycobacteriosis in assays (Table 3), the CBPR and Idaho pygmy rab- pygmy rabbits are nonspecific, the high incidence bits responded to both mitogen (phytohemaggluti- of mycobacteriosis in the population suggests my- nin, concanavalin A, and pokeweed mitogen) and cobacteriosis should be considered in any pygmy allogeneic mixed lymphocyte antigens (major his- rabbit with clinical signs of illness. The nonspecific tocompatibility complex) to significantly lesser de- hematologic and biochemistry panel abnormalities grees than did either domestic rabbits or intercross of heterophilia, monocytosis, anemia, hypoalbu- pygmy rabbits. None of the pygmy rabbits respond- minemia, hyperglobulinemia, and/or hypercholes- ed to the bovine derivative of mycobacteria (bacille terolemia are common findings in clinical cases. Calmette-Gue´rin, BCG) or to the saprophytic my- Diagnosis required sampling of masses, tissues, cobacteria, M. vaccae. However, the CBPR and or excreta (blood, urine, or feces) for discovery of Idaho pygmy rabbits, as well as the intercross pyg- acid-fast positive bacilli through cytologic prepa- my rabbits, responded to M. avium. This positive ration or culture. Urine or blood mycobacterial cul- response to the M. avium antigen did not depend tures were more sensitive than fecal mycobacterial on the clinical status of the individual rabbit. culture. Several sites should be sampled, because Pygmy rabbits produced significantly less gam- individual patients may have mycobacterial organ- ma-interferon and more interleukin-10 than did do- isms in only one particular organ system; serial mestic rabbits (Table 4). They produced lower con- sampling also should be considered, because de- centrations of tumor necrosis factor, interleukin-1, tectable shedding may wax and wane during the and interleukin-6, but more interleukin-2 than do- disease course.38 Variable shedding over time and/ mestic rabbits. Specifically, T helper 1 (Th1) cy- or suboptimal sensitivity of initial mycobacterial 506 JOURNAL OF ZOO AND WILDLIFE MEDICINE b 10 for the Idaho CBPR Idaho CBPR Idaho CBPR Idaho CBPR CBPR Idaho CBPR Idaho CBPR Intercross Idaho CBPR Idaho CBPR Idaho CBPR Idaho CBPR Idaho CBPR Intercross Idaho CBPR 0.01 Ͼ Ͼ Ͼ Ͼ Ͼ Ͼ Ͼ Ͼ Ͼ Ͻ Ͻ Ͻ Ͻ Ͻ Ͻ Ͼ Ͼ Ͼ Ͼ Ͼ Ͼ Ͼ Ͼ Ͼ Ͻ Ͻ Ͻ Ͻ Ͻ CBPR CBPR CBPR CBPR CBPR P Significant differences, Ͼ Ͼ Ͼ Ͻ Ͻ Sylvilagus bachmani Intercross Intercross Idaho Intercross Idaho Intercross Idaho Idaho Intercross Idaho Intercross Domestic Domestic Intercross Domestic Intercross Domestic Intercross Domestic Intercross Domestic Intercross Domestic Domestic Intercross 16.8 Domestic 14.4 Domestic 14.3 Domestic 28.6 Domestic 0.20.3 Intercross Domestic 0.2 Domestic 5 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ ϭ n Control– Domestic, 1 SD). A stimulation index value of Ϯ a 1 ϭ Brush, n Control– ) of Columbia Basin subpopulation origin (CBPR), adult pygmy rabbits of 4.7 159.35.4 173.9 141.65.7 143.7 150.24.9 162.0 88.50.4 120.6 0.5 1.3 0.50.4 1.6 1.0 0.2 0.5 7 0.01. Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ ϭ Ͻ n P Intercross, Control-Domestic). Each data point is presented as a ‘‘stimulation index’’ value, which is Brachylagus idahoensis 11.6 96.3 10.4 126.0 6.8 155.9 10.4 86.0 0.30.4 1.1 0.9 2.3 2.2 15 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ ϭ Idaho, n Oryctolagus cuniculus, -test at the significance threshold of t 4.5 68.4 4.8 88.3 4.5 133.5 6.5 50.5 0.40.5 1.3 1.7 2.0 9.5 18 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ ϭ CBPR, n 2.2 10.8 ´rin 1.7 3 for the antigens is considered positive. Ͼ Lymphocyte stimulation indices of adult pygmy rabbits ( Test Control-Brush), and adult domestic rabbits ( Values were compared between groups using Student’s Values for the California riparian brush rabbit were not statistically evaluated because the sample size was one. Table 3. a b Idaho subpopulation origin (Idaho), adult pygmy rabbits of CBPR–Idaho intercross origin (Intercross), an adult California riparian brush rabbit ( riparius, mitogens and the ratio of counts per minute in the stimulated culture divided by a parallel unstimulated control culture (mean Phytohemagglutinin 66.8 Pokeweed mitogenMixed lymphocyte culture 126.9 Bacille 41.2 Calmette-Gue Mycobacterium vaccae Mycobacterium avium Concanavalin A 78.5 HARRENSTIEN ET AL.—MYCOBACTERIUM AVIUM IN PYGMY RABBITS 507 Control– b Idaho CBPR CBPR Idaho CBPR Idaho CBPR Idaho CBPR Idaho CBPR CBPR CBPR Intercross CBPR CBPR CBPR Idaho CBPR 0.01 Ͼ Ͼ Ͼ Ͻ Ͻ Ͻ Ͻ Ͻ Ͻ Ͻ Ͼ Ͻ Ͼ Ͻ Ͼ Ͼ Ͻ Ͼ Ͻ Ͼ CBPR CBPR CBPR CBPR CBPR CBPR CBPR P Significant differences, Ͼ Ͼ Ͻ Ͼ Ͻ Ͼ Ͼ Intercross Idaho Idaho Intercross Idaho Idaho Idaho Intercross Idaho Intercross Idaho Intercross Intercross Domestic Intercross Intercross Intercross Domestic Intercross Sylvilagus bachmani riparius, 12.2 Intercross 4.05.1 Domestic Domestic 2.6 Domestic 1.11.7 Domestic Domestic 4.2 Domestic 5 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ ϭ n Control– Domestic, a 1 ϭ Brush, n Control– 2.3 121.53.9 117.0 4.8 21.5 24.3 19.5 1.7 22.1 14.60.8 14.3 1.8 8.5 16.24.0 6.2 12.3 23.8 23.3 7 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ ϭ n 0.01, except for the California riparian brush rabbit’s values, which were not statistically Intercross, Ͻ P ) of Columbia Basin subpopulation origin (CBPR), adult pygmy rabbits of Idaho subpopu- 4.211.0 111.1 5.9 23.2 23.1 10.9 5.7 2.96.9 5.5 20.2 9.4 22.7 15 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ ϭ Idaho, n Control–Domestic). Brachylagus idahoensis 3.30.3 106.3 5.3 24.0 30.8 0.5 20.3 2.00.5 7.1 15.2 0.6 39.9 -test at the significance threshold of t 18 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ ϭ CBPR, n Oryctolagus cuniculus, (pg/ml) 8.1 ␣ Test Cytokine profiles of adult pygmy rabbits ( Values were compared between groups using Student’s Values for the California riparian brush rabbit were not statistically evaluated, because the sample was 1. Table 4. a b lation origin (Idaho), adultBrush), pygmy and rabbits adult of domestic CBPR–Idaho rabbits intercross ( origin (Intercross), an adult California riparian brush rabbit ( Complement CH50 (pg/ml) 101.8 Interleukin-1 (pg/ml)Interleukin-2 (pg/ml)Interleukin-6 (pg/ml) 2.7 Interleukin-10 (pg/ml) 46.2 Gamma-interferon (pg/ml) 4.1 Tumor necrosis factor- 12.7 2.8 compared due to a sample size of 1. 508 JOURNAL OF ZOO AND WILDLIFE MEDICINE cultures could be responsible for the conversion of narians and facility managers, related to individual some rabbits from culture-negative to culture-pos- case circumstances and to determination of treat- itive status during the course of antibiotic therapy. ment benefits and risks. Because captive reproduc- The long interval between sample collection and tion is critical to the CBPR endangered species re- generation of results (up to 6 wk in some cases) covery project, current recommendations include interfered with prompt diagnosis of mycobacterio- treatment and attempted cure and/or control of M. sis in pygmy rabbits; PCR technology should be avium–infected breeding animals. Concerns were used more extensively to reduce diagnostic turn- raised initially regarding the tolerance of these shy around time.19 animals for twice-daily manual restraint for admin- One rabbit of this series grew M. avium scrofu- istration of medication. Body weights, fecal char- laceum in repeated cultures of urine and blood; cul- acter, and mentation appeared unchanged through- ture isolates were initially PCR-negative for M. tu- out the course of therapy in several animals that berculosis and M. avium and further testing clari- were monitored closely for adverse effects. Pygmy fied the species identity. This finding underscores rabbits that were transferred from soil enclosures to the importance of full speciation of acid-fast bacilli standard laboratory metal rabbit hutches for disease in cultures from patients. treatment appeared to adapt to the provided artifi- Intradermal tuberculin testing was considered as cial systems. As of this writing, one M. av- a possible surveillance tool for pygmy rabbits, but ium culture-positive rabbit has received more than was not investigated. If employed, exposure of pyg- 6 mo of continuous azithromycin-ethambutol-rifa- my rabbits to M. avium antigens via intradermal butin treatment without visible adverse effects. testing may have sensitized them and caused later Tree kangaroos housed without soil substrate do antibody assays to be invalid.7 not appear to develop disseminated mycobacterial After initial thoracic, abdominal, and skeletal ra- infections, in contrast to the many M. avium cases diographs were carefully evaluated, further radio- noted in tree kangaroos with soil access (M. Bush, graphic evaluation did not appear necessary, be- pers. comm.). Housing of captive pygmy rabbits cause the presence and character of radiographic without soil substrate is being investigated as a hus- lesions did not change over the 1–3 mo course of bandry measure for mycobacteriosis prevention. disease. Given the prevalence of bone colonization, Lymphocyte stimulation and cytokine assays it is possible that advanced diagnostics such as were helpful in explaining the high morbidity and scintigraphy may detect early osteomyelitis cases.37 mortality associated with mycobacterial infection in Captive facilities have adopted postmortem sam- pygmy rabbits. Cell-mediated immune responses pling protocols that require histopathology and my- are necessary for clearance of mycobacterial infec- cobacterial testing of all dead adult animals. Re- tion in healthy animals. Mononuclear cell responses cently, a case of mycobacterial infection without to plant mitogens and major histocompatibility granuloma formation was found in a wild-caught complex antigens give an assessment of total cell- male of Idaho population origin that had been in mediated immune response, because these agents captivity at OZ and WSU for 17 mo. The rabbit stimulate a large fraction of mononuclear cells. The was euthanized due to chronic weight loss; post- control rabbits responded more vigorously to mi- mortem tissue cultures were positive for M. avium, togens and major histocompatibility complex anti- but histologic evidence of granuloma formation gens than did the CBPR and Idaho pygmy rabbits, was not seen. If more extensive antemortem and as shown by higher stimulation indices. The re- postmortem sampling had been performed or if a duced responses of CBPR and Idaho pygmy rabbits longer time interval before euthanasia had tran- may be due to multiple factors such as environ- spired, granulomas may have been found. In fact, ment, genetics, and external stressors. In contrast, immunoblot data from serum collected at time of the lymphocyte responses of the intercross pygmy euthanasia from this animal showed a high anti- rabbits to mitogens were similar to those of the do- body response to M. avium. Nonfatal mycobacterial mestic rabbits, suggesting a possible ‘‘hybrid vig- infection has been reported in marsupials.7 or’’ benefit of better cell-mediated immunity in Given the high morbidity and mortality associ- these intercrossed animals. As individuals of the in- ated with M. avium in pygmy rabbits, pharmaceutic tercross pygmy rabbit population become older, it treatment should be considered as soon as acid-fast is theorized that they will show a lower incidence bacilli are seen or a positive mycobacterial culture of fatal mycobacteriosis than do CBPR or Idaho is obtained. M. avium is not a public health concern pygmy rabbits. except for immunocompromised persons; therefore, All of the pygmy rabbits responded to the M. treatment decisions remain in the hands of veteri- avium antigen, but not the BCG or M. vaccae. The HARRENSTIEN ET AL.—MYCOBACTERIUM AVIUM IN PYGMY RABBITS 509 uniform response to M. avium suggests that ex- clinical debate. Human peak serum values of these posed pygmy rabbits could mount some degree of drugs are seen at 2–3 hr after oral administration, response to the presented antigen. However, their therefore, pygmy rabbits were sampled during this response did not lead to effective control and/or period; peak values, however, may have been killing of the M. avium organism, a situation often achieved at different times in the pygmy rabbits. observed in species with impaired immune sys- Domestic rabbits receiving the same drug dosages tems.8,25,27,34 For this reason, mycobacterial vacci- as the pygmy rabbits of our study have shown peak nation of pygmy rabbits is not predicted to be of serum values that correspond well with recom- clinical benefit for disease prevention. mended human peak serum concentrations, but Under the influence of cytokines, T helper cells peak concentrations in domestic rabbits were seen can differentiate either into Th1 cells, which induce at times other than 2–3 hr after administration: a cytotoxic, cellular immune response, or into Th2 azithromycin at approximately 1 hr postadministra- cells, which induce a humoral response. Therefore, tion, rifabutin at 4 hr postadministration, and eth- the Th1/Th2 differentiation state of T helper cells ambutol at 0.5 hr postadministration.2,11,36 Due to can be assessed by differential cytokine production. sparse drug concentration sampling performed in Compared with domestic rabbits, the pygmy rabbits this study, definitive conclusions regarding the bio- demonstrated lower expression of interleukin-1, in- availability and potential clinical utility of the drugs terleukin-6, and gamma-interferon and elevated ex- tested cannot be drawn. Based on available data pression of interleukin-10. These data imply that from this report, azithromycin (50 mg/kg p.o. q 24 they are mounting a Th2, or humoral, type of im- hr) and rifabutin (25 mg/kg p.o. q 12 hr) appear to mune response. Immunoblot and/or enzyme-linked be viable options for the treatment of pygmy rab- immunosorbent assay (ELISA) testing of serum or bits, but the dosing of rifampin and ethambutol re- plasma for antibody relies upon presence of a hu- quire additional study before each can be recom- moral response to M. avium exposure. Specific an- mended with confidence.28,29 Preliminary evidence tibody responses to M. avium were detected by im- suggests that ethambutol may be a useful adjunct munoblot in all of the tested pygmy rabbits of this therapy at 45 mg/kg p.o. q 12 hr as part of triple study, and preliminary ELISA values show strong combination therapy. Additional testing is required correlation with immunoblot data (Paustian, un- to fully describe the pharmacokinetics of these publ. data). Assuming mycobacterial exposure re- drugs in pygmy rabbits. sults in high mortality in pygmy rabbits, antibody Several adjunct therapies have been investigated testing may be a valuable means of early detection for M. avium disease in other species and may show of those rabbits considered candidates for myco- promise in the future for pygmy rabbits as well, bacterial treatment. pending investigation of appropriate administration A Th1 response, or cytotoxic cellular response, logistics. Synthetic gamma-interferon-1-beta (Ac- is required to clear mycobacterial infections. Th1 timmune௡, Intermune, Inc., Brisbane, California responses are inhibited by high amounts of Th2 cy- 94005 USA) is often recommended as adjunct ther- tokines. The pygmy rabbit Th1/Th2 response was apy for humans with M. avium–associated disease. imbalanced. Gamma-interferon concentrations were Although Th1 response depression in humans is reduced, and therefore, macrophage activation was due typically to retroviral infection, rather than in- decreased. Under such circumstances, low numbers herent cell-mediated immunity depression as sus- of killer T cells also would be expected. Humans pected in pygmy rabbits, it is possible that pygmy with clinical mycobacteriosis typically have low rabbits with mycobacteriosis could benefit from gamma-interferon production.46 A Th2 polarization gamma-interferon therapy. Granulocyte-macro- is found in some strains of mice, associated with phage colony-stimulating factor has been found to multiple genetic loci that control susceptibility to be synergistic with azithromycin in the treatment of intracellular infections.4,12,35 It is likely that the murine M. avium infections and may be synergistic same types of multigeneic differences induce a Th2 with azithromycin in pygmy rabbits, as well.3 Other response in pygmy rabbits leading to disseminated adjunct therapies may be warranted, including vi- infection, rather than a protective Th1 response. tamin E and/or nonsteroidal anti-inflammatory Humans with M. avium infection typically are medications for relief of tissue inflammation and treated with a three-drug combination consisting of discomfort. Mycobacterial colonization may occur a macrolide (azithromycin or clarithromycin), eth- more readily in traumatized or inflamed tissue than ambutol, and rifabutin (or rifampin).10 The use of in healthy tissue,45 therefore, reduction of tissue in- drug susceptibility testing to guide the treatment of flammation might reduce the number of possible mycobacteriosis in humans currently is subject to foci of granuloma formation and make systemic 510 JOURNAL OF ZOO AND WILDLIFE MEDICINE mycobacterial clearance more effective. Once- or for provision of a riparian brush rabbit blood sam- twice-weekly azithromycin administration is rec- ple, and Dr. Kristin Kemper, Dr. Kristin Gunderson, ommended for at-risk human patients (HIV-infect- Margot Monti, and Dr. Keith Riley for their help ed, with fewer than 50 CD4 T-lymphocytes per ␮l summarizing and interpreting early data. Manu- of peripheral blood),10 and prophylactic therapy is script drafts were reviewed by David Hays and Dr. under investigation for pygmy rabbits at our facil- Kenneth Warheit of the Washington Department of ities, as well. Fish and Wildlife.

CONCLUSIONS LITERATURE CITED The pygmy rabbits examined in this study exhib- 1. Bannantine, J., and J. R. Stabel. 2000. HspX is pres- ited partially ineffective cell-mediated immunity, ent within Mycobacterium paratuberculosis-infected mac- which we believe predisposed them to morbidity rophages and is recognized by sera from some infected and mortality from M. avium complex infection. cattle. Vet. Microbiol. 76: 343–358. Physical examination, acid-fast staining of cytolog- 2. Battaglia, R., E. Pianezzola, G. Salgarollo, G. Zini, ic preparations of directed samples (aspirates or im- and M. Strolin-Benedetti. 1990. Absorption, disposition pression smears of masses), thoracic and skeletal and preliminary metabolic pathway of 14C-rifabutin in animals and man. J. Antimicrob. Chemother. 26: 813–822. radiography, hematologic and serum chemistry 3. Bermudez, L. E., J. Martinelli, M. Petrofsky, P. Ko- analysis, and serum/plasma antibody assays appear lonosky, and L. S. Young. 1994. Recombinant granulo- to be useful tools for presumptive diagnosis of my- cyte-macrophage colony-stimulating factor enhances the cobacteriosis in pygmy rabbits, allowing early med- effects of antibiotics against Mycobacterium avium com- ical treatment and a potential cure. Mycobacterial plex infection in the beige mouse model. J. Infect. Dis. culture of blood, urine, feces, and/or directed sam- 169: 575–580. ples is a specific technique for diagnosis, but the 4. Bix, M., and R. M. Locksley. 1998. Independent and low sensitivity and long time required for growth epigenetic regulation of the IL-4 alleles in CD4ϩ T cells. of mycobacteria make culture less valuable for ini- Science 281: 1352–1354. tial clinical assessments and more valuable for 5. Buddle, B. M., and L. J. Young. 2000. Immunobi- ology of mycobacterial infections in marsupials. Dev. long-term confirmation of diagnosis. Pygmy rabbits Compar. Immunol. 24: 517–529. with M. avium infection should be treated with 6. Burns, D. L., R. S. Wallace, and J. A. Teare. 1994. azithromycin (50 mg/kg p.o. q 24 hr) and rifabutin Successful treatment of mycobacterial osteomyelitis in a (25 mg/kg p.o. q 12 hr). Failure to eliminate my- Matschie’s tree kangaroo (Dendrolagus matschiei). J. Zoo cobacterial infections in these pygmy rabbits likely Wildl. Med. 25: 274–280. was related to advanced infection status at diag- 7. Bush, M., and R. J. Montali. 1998. Medical man- nosis and/or poor drug selection in some of the cas- agement of tree kangaroos. In: Fowler, M. E., and R. E. es. Mycobacteriosis prevention in pygmy rabbits Miller (eds.). Zoo and Wild Animal Medicine, 4th ed. W. should include nonsoil housing or use of presteril- B. Saunders Co., Philadelphia, Pennsylvania. Pp. 337– ized soil in enclosures, exclusion of carrier birds 343. 8. Bush, M., R. J. Montali, S. Murray, S. Holland, and from enclosures, and improvement of cell-mediated T. M. Phillips. 1995. The diagnosis, treatment and pre- immunocompetence of the population, which might vention of tuberculosis in captive Matschie’s tree kanga- be achieved through intercross breeding of animals roos (Dendrolagus matschiei). Proc. Am. Assoc. Zoo Vet., of CBPR and Idaho subpopulation origins. Wildl. Dis. Assoc., Am. Assoc. Wildl. Vet. 1995: 312–314 (Abstr.). Acknowledgments: We thank the staff, graduate 9. Campbell III, T. M., T. W. Clark, and C. R. Groves. students, and volunteers of the Oregon Zoo, Wash- 1982. First record of pygmy rabbits (Brachylagus ida- ington State University, and the Washington De- hoensis) in Wyoming. Great Basin Nat. 42: 100. partment of Fish and Wildlife for their hard work 10. Centers for Disease Control. 2002. Guidelines for caring for these pygmy rabbits and maintaining preventing opportunistic infections among HIV-infected their medical records. Special thanks go to Jan persons, 2002. Morb. Mortal. Wkly. Rep. 51(RR-8): 10– Steele, Michael Illig, and Dr. Lisa Shipley for man- 11. Available at: http://www.cdc.gov/mmwr/PDF/rr/ agement of the captive breeding programs. Captive rr5108.pdf. Accessed on 25 October 2004. 11. Chen, M. M., C. S. Lee, and J. H. Perrin. 1984. management of pygmy rabbits is supported in part Absorption and disposition of ethambutol in rabbits. J. by the Washington Department of Fish and Wildlife Pharm. Sci. 73: 1053–1055. and the United States Fish and Wildlife Service and 12. Coffman, R. L., and A. M. Beebe. 1998. Genetic is managed by David Hays and Chris Warren. The control of the T cell response to Leishmania major infec- authors also thank Dr. Kirsten Gilardi of the Uni- tion. Adv. Exp. Med. Biol. 452: 61–66. versity of California, Davis, Wildlife Health Center, 13. Daniels, M. J., M. R. Hutchings, P. M. Beard, D. HARRENSTIEN ET AL.—MYCOBACTERIUM AVIUM IN PYGMY RABBITS 511

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