Disseminated Mycobacterium celatum infection in a white-tailed trogon ( Trogon viridis )

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Disseminated Mycobacterium celatum infection in a white-tailed trogon (Trogon viridis)

Journal: Avian Pathology

Manuscript ID: CAVP-2005-0012.R3

Manuscript Type: Original Research Paper

Date Submitted by the Author:

09-Apr-2006

Complete List of Authors: Bertelsen, Mads; Copenhagen Zoo, Center for Zoo and Wild Animal Health Grøndahl, Carsten; Copenhagen Zoo, Center for Zoo and Wild Animal Health Giese, Steen; Danish Institute for Food and Veterinary Research

Keywords: Mycobacteriosis, Mycobacterium celatum, Trogon viridis, granulomatous inflammation

E-mail: [email protected] URL: http://mc.manuscriptcentral.com/cavp

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Cavp-2005-0012

Disseminated Mycobacterium celatum infection in a white-tailed trogon (Trogon

viridis)

Mads F. Bertelsen1*

, Carsten Grøndahl1 & Steen B. Giese

2

1Center for Zoo and Wild Animal Health, Copenhagen Zoo, Sdr. Fasanvej 79, DK-2000

Frederiksberg, Denmark, 2

Danish Institute for Food and Veterinary Research, Bülowsvej 27, DK-

1790 København V, Denmark

Running title: Mycobacterium celatum in a trogon

* To whom correspondence should be addressed.

Tel +45 7220 0262 Fax: +45 7220 0264 E-mail: [email protected]

Received: 16 December 2005

NOTE: AUTHOR WISHES FIGURES TO BE IN BLACK & WHITE PRINTED VERSION AND

COLOUR ONLINE – IMAGES SHOULD BE PRINTED 8CM WIDE.

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Cavp-2005-0012

Disseminated Mycobacterium celatum infection in a white-tailed trogon (Trogon

viridis)

Mads F. Bertelsen1*

, Carsten Grøndahl1 & Steen B. Giese

2

Abstract:

An adult female white-tailed trogon (Trogon viridis) was presented with abdominal enlargement

and hard subcutaneous masses. Necropsy findings included bony masses extending from skeletal

structures, disseminated pale foci in the liver, and a pale mass in the kidney. Histological

examination revealed multifocal to coalescing granulomatous inflammation in bone, liver, kidney,

lung and spleen. Mycobacterium celatum was isolated from the liver and identified by DNA

sequencing. This is the first report of M. celatum infection in an avian species.

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Introduction

Mycobacterium celatum is a slowly growing, potentially pathogenic mycobacterium (Tortoli, 2003),

first described in 1993 (Butler et al. 1993). M. celatum is divided into three groups (types 1, 2 and

3) based on genomic sequencing (Butler et al. 1993, Bull et al. 1995). Originally reported to cause

infection mainly in AIDS patients (Piersimoni et al. 1994, Tortoli et al. 1995, Bonomo et al. 1998),

the agent has recently been identified as the cause of serious, often disseminated, infection in

immunocompetent individuals (Piersimoni et al. 2003). The only report of M. celatum occurring

naturally in an animal is that of an adult male domestic ferret with severe disseminated

granulomatous inflammation (Valheim et al. 2001).

Avian mycobacteriosis is most commonly caused by Mycobacterium avium, M.

intracellulare and M. genavense, while infections with M. tuberculosis, M. fortuitum, and M. bovis

occur relatively infrequently (Hoop et al. 1996, Tell et al. 2001).

We report a case of M. celatum in a trogon. To the authors’ knowledge this is the first

description of infection with M. celatum in an avian species.

Materials and Methods

Case history. An approximately 4-year-old white-tailed trogon (Trogon viridis) was presented for

clinical examination in March 2005, with a history of impaired flight and soiling of the beak with

feed materials. The bird was hatched and reared in a German facility and was housed in a large free

flight aviary in Denmark since October 2003.

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At presentation the bird was alert, the body condition was good, and the plumage was in

excellent condition. Below the mandible, there was a hard spherical (15 mm in diameter) bony

mass, and a similar but larger (25 mm in diameter) mass was present subcutaneously on the left

wing, immediately distal to the elbow. The cranial abdomen was markedly distended. Based on the

history and clinical presentation the prognosis was considered poor, and the bird was euthanized.

Pathology. The submitted bird was weighed and examined. Representative samples of liver,

kidney, adrenal, heart, lung, spleen, pancreas, oesophagus, proventriculus, ventriculus, small and

large intestine, bone, skin, and skeletal muscle were collected in 10% neutral buffered formalin,

processed routinely for histopathology, and embedded in paraffin blocks. Sections 4 to 7 µm thick

were cut, mounted on glass slides, and stained with haematoxylin and eosin (HE), and with Ziehl-

Neelsen stain (ZN).

Immunohistochemical analysis for mycobacteria was performed on paraffin-embedded

sections of the liver using an anti Mycobacterium bovis rabbit polyclonal antiserum and a standard

avidin-biotin complex immunohistochemical method at Prairie Diagnostic Services, Western

College of Veterinary Medicine, Saskatoon, Saskatchewan, Canada, as previously described

(Haines & Chelack 1991, Plante et al. 1996). Briefly, tissue sections were deparaffinized and

rehydrated. Endogenous tissue peroxidases were inactivated by immersion of the slides in a solution

of 0.5% H202 in methanol for 30 min at room temperature. Sections were then digested by

immersion in a solution of protease XIV (Sigma Chemical, St Louis, Missouri, USA) 0.5 mg/mL in

0.1M; pH = 7.4 phosphate buffered saline (PBS) (prewarmed to 37°C) for 15 min at 37°C, and

washed 3 times in PBS for 5 min. Nonspecific adherence of proteins to tissue sections was saturated

by a 30 min room temperature incubation of sections flooded with PBS supplemented with 4.0%

normal rabbit serum. The saturating solution was drained from the slides and the antiserum (M.


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bovis #B124, Dako Corp, Via Real, Carpinteria, California, USA), diluted 1/2000 and 1/4000 in

PBS containing 4.0% normal rabbit serum, was applied to duplicate sections. The slides were

incubated overnight at 4°C then washed 3 times in PBS for 5 min and biotinylated antibody to

rabbit immunoglobulin (Vector Laboratories) was applied diluted 1/200 in PBS containing 4.0%

normal rabbit serum. The slides were incubated for 30 min at room temperature, then washed 3

times in PBS for 5 min. The avidin-biotin complex peroxidase solution was prepared according to

the manufacturer's directions and incubated on the tissue sections for 30 min at room temperature,

then the slides were washed 3 times in PBS for 5 min. The sections were exposed to the chromagen,

3,3 diaminobenzadine-4HCl (DAB, Electron Microscopic Products, Fort Washington,

Pennsylvania, USA) 1 mg/mL in PBS supplemented with H202 (10 uLL of 50% H202 in 5 mL

PBS), incubated at room temperature for 5 min, washed 3 times in PBS for 5 min and

counterstained with Mayers hematoxylin. Immunohistochemical analysis was not performed on

other organs.

Microbiology. Liver tissue was cultured for mycobacteria at the Danish Institute for Food and

Veterinary Research. The tissue was decontaminated with 2 ml 5% sulphuric acid and put in a

stomacher for 1 minute. After an additional 10 minutes, 10 ml of saline (0.9 %) was added and the

sample was mixed and centrifuged for 10 minutes at 1000 g. The supernatant was discarded and the

sediment re-suspended and cultured on Löwenstein-Jensen media. The tubes were incubated

aerobically at 37°C and read for growth every second week for two months.

Isolates were analysed using standard identification kits (Gen-Probe, San Diego, CA,

USA), and identification was subsequently performed by genome sequencing as previously

described (L’Abée-Lund et al. 2003). Bacteria from one colony were suspended in distilled water

and lysed by boiling. The rDNA was PCR amplified for 35 cycles at 940 C for 1 min., 550 C for 1

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min. and 720 C for 1.5 min. using primers 8FX and 1509RX (see below). The amplification

products were purified with QIAqiuck® spin columns(QIAgen, Hilden, Germany) and sequenced

on an ABI 377 automatic sequencer using the PRISM® BigDye Terminator cycle sequencing kit

(Applied Biosystems, Foster City, CA, USA) and the following primers: 8FX: 5-

'AGAGTTTGATCCTGGCTNAG-3' ; 517F: 5'-CCAGCAGCCGCGGTAATAC-3';535R:5'-

GTATTACCGCGGCTGCTGG-3' ; 784FX:5'-GATTAGATACCCNGGTAGTC-3' ; 804 RX: 5'-

GACTACCNGGGTATCTAATCC-3' ; 1407R: 5'-TGACGGGCGGTGTGTACAA-3' ;1509RX: 5’-

GTTACCTTGTTACGACTTCAC-3’. All primers are universal (Weisburg et al. 1991) allowing

sequencing of both strands. The numbering of the primers refers to the numbering of E. coli 16S

rDNA (Brosius et al. 1978).

Results

At necropsy, the trogon was found to be in good body condition. Body weight was 85g.

Subcutaneously, below the mandible, adhered to the right hyobranchial apparatus was a roughly

spherical, multilobulated bony mass (Figure 1).. On the cut surface, the mass was found to be

heavily mineralised and traversed by bony trabeculae. On the left wing immediately distal to the

elbow a similar mass measuring 25 mm in diameter was present, extending from the ulnar cortex.

The liver was enlarged, with markedly rounded edges. Scattered randomly throughout the organ

were multiple 0.5 to 2 mm pale foci. Near the caudal pole of the right kidney was a 2 x 3 x 3 mm

pale yellow mass. The spleen was pale, but of normal size and texture. There were no significant

findings in other organs..

Histopathological examination of the liver showed multiple, randomly scattered granulomas of 100-

500 µm consisting of a thick loosely woven capsule of whirling spindle-shaped fibrocytes

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surrounding a centre of relatively small macrophages and occasional multinucleated giant cells

(Figure 2). In several granulomas, a central area of caseous necrotic debris was surrounded by a

mantle of multinucleated giant cells. A very low number of acid-fast organisms were identified.

Similar changes were observed in the spleen, where multiple, coalescing granulomas of 200-400

µm characteristically lacked areas of central necrosis, but contained centres of macrophages, some

of which had a finely granular (ground glass) cytoplasm. In ZN stained sections, a low number of

acid fast organisms were demonstrated. In the lung, a large (appr. 1 x 1 mm), and several smaller

granulomas were present near one of the parabronchi. Here a thin (30-50 µm) mantle of fibrocytes

surrounded a layer of multinucleate giant cells walling off a large centre of necrotic debris and

occasional acid fast organisms (Figure 3). The kidneys had occasional small (200-300 µm)

granulomas characterized by a central area of necrosis walled off by a mantle of multinucleated

giant cells, as well as a very large (5 mm) granuloma containing several large necrotic foci. No acid

fast organisms were identified in these lesions. The bony lesions were characterised by large masses

of cancellous bone comprised bony trabeculae, arising from the original cortex, intermixed with

islets of normally appearing bone marrow and scattered large granulomas (300 µm – 5mm), often

containing large necrotic areas (Figure 4). As in other organs, ZN stained sections revealed low

numbers of acid fast organisms.

Massive amounts of mycobacterial antigen was demonstrated by immunohistochemical

analysis of liver sections.

Mycobacterial growth was detected after 2 weeks of incubation. The isolate gave reactions

below cut-off with the AccuProbe MAC (M. avium complex), the AccuProbe MTB (M. tuberculosis

complex) and the AccuProbe MAV (M. avium) identification kits (Gen-Probe, San Diego, CA,

USA).


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Sequence data when compared to 16S sequences in Genebank demonstrated that the

isolate had a 16S rDNA sequence identical to that of M. celatum type 1 (EMBL L08169, Bull et al.

1995).

Discussion

Based on the findings described above, M. celatum was considered to be the etiological agent of the

severe disseminated granulomatous inflammation in this trogon. The classical tuberculous lesions

and well-defined granulomas observed in the trogon are in contrast to the poorly defined

granulomas described in humans (e.g., Piersimoni et al. 1994, Bonomo et al. 1998) and a ferret

(Valheim et al. 2001) infected with M. celatum. Indeed, the lesions present in the trogon are

identical to those seen in classical avian mycobacteriosis caused by M. avium (Montali et al. 1976,

Tell et al. 2001, Schmidt et al. 2003). Bony involvement was a characteristic feature in the present

case, as has been described with varying frequency in birds infected with other mycobacterial

agents (Montali et al. 1976, Tell et al. 2001). Compared to the massive antigen presence

demonstrated immunohistochemically, relatively low numbers of organisms were identified in ZN-

stained sections. As acid-fast staining generally detects intact mycobacterial cells, a possible reason

for this could be that the immunohistochemical staining is identifying structurally incomplete

bacteria.

The route of infection of M. celatum is poorly known, but the presence of pulmonary

lesions in the absence of granulomas in the intestinal tract in this trogon may suggest transmission

by inhalation, as was proposed in a ferret (Valheim et al. 2001). In such case M. celatum would

exhibit a pattern similar to that of M. tuberculosis and be in contrast to the typically intestinal origin

of infections with M. avium and M. genevense (Tell et al. 2001).


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The M. celatum isolate from this case could not be distinguished from the EMBL M.

celatum type 1 strain (Bull et al. 1995), and the source of infection in this case was unknown. Most

mycobacteria are ubiquitous environmental saprophytes (Grange et al. 1990) and although isolation

of M. celatum from the environment has not yet been reported (Tortoli, 2003), exposure from soil or

water remains the most probable source. The trogon was hatched and reared in a German facility

and had been at the zoo for 17 months when it presented with clinical signs. Considering the

chronic and insidious nature of the mycobacterial infections, exposure at either institution is

possible. Little is known about the abundance of M. celatum. Currently approximately 3 cases of

human infection are seen annually in Denmark. (Z. Kamper-Jørgensen, personal communication)

This case report demonstrates that M. celatum must be added to the list of agents causing

mycobacteriosis in birds. The pathogenicity and importance of this agent remain to be investigated.

Acknowedgement

The authors thank Dr BrianChelack for the description of the IHC methodology.


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References

L’Abée-Lund, T.M., Heiene, R., Friis, N.F., Ahrens, P. & Sørum, H. (2003). Mycoplasma canis and

urogenital disease in dogs in Norway. Veterinary Record 153, 231-235.Bonomo, R.A.,

Briggs, J.M., Gross, W., Hassan, M., Graham, R.C., Butler, W.R. & Salata, R.A. (1998).

Mycobacterium celatum infection in a patient with AIDS. Clinical Infectious Diseases, 26,

243-245.

Brosius, J., Palmer, M.L., Kennedy, P.J. and Noller, H.F. (1978). Complete nucleotide sequence of

a 16S ribosomal RNA gene from Escherichia coli. Procedings of the National Academy of

Science, USA, 75, 4801-4805.

Bull, T.J., Shanson, D.C., Archard, L.C., Yates, M.D., Hamid, M.E. & Minnikin, D.E. (1995). A

new group (Type 3) of Mycobacterium celatum isolated from AIDS patients in the London

area. International Journal of Systematic Bacteriology, 45, 861-862.

Butler, W.R., O’Connor, S.P., Yakrus, M.A., Smithwick, R.W., Plikaytis, B.B., Moss, C.W., Floyd,

M.M., Woodley, C.L., Kilburn, J.O., Vadney, F.S. & Gross, W.M. (1993). Mycobacterium

celatum sp. nov. International Journal of Systematic Bacteriology, 43, 539-548.

Grange, J.M., Yates, M.D. & Boughton, E. (1990). The avian tubercle bacillus and its relatives.

Journal of Applied Bacteriology, 68, 411-431.

Haines, D.M. & Chelack, B.J. (1991). Technical considerations for developing enzyme

immunohistochemical staining procedures on formalin-fixed paraffin-embedded tissues for

diagnostic pathology. Journal of Veterinary Diagnostic Investigation, 3, 101-112.

Hoop, R.K., Böttger, E.C. & Pfyffer G.E. (1996). Etiological agents of mycobacteriosis in pet birds

between 1986 and 1995. Journal of Clinical Microbiology, 34, 991-992.

Montali, R.J., Bush, M., Thoen, C.O. & Smith, E. (1976). Tuberculosis in captive exotic birds.

Journal of the American Veterinary Medical Association, 169, 920-927.

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Piersimoni, C., Tortoli, E. & De Sio, G. (1994). Disseminated infection due to Mycobacterium

celatum in a patient with AIDS. Lancet, 344, 332.

Piersimoni, C., Zitti, P.G., Nista, D. & Bornigia, S. (2003). Mycobacterium celatum Pulmonary

infection in the immunocompetent: Case report and review. Emerging Infectious Diseases, 9,

399-402.

Plante, Y., Remenda, B.W., Chelack, B.J. & Haines D.M. (1996). Detection of Mycobacterium

paratuberculosis in formalin-fixed paraffin-embedded tissues by the polymerase chain

reaction. Canadian Journal of Veterinary Research. 60,115-120

Schmidt, R.E., Reavill, D.R. & Phalen, D.N. (2003). Liver. In Schmidt, R.E., Reavill, D.R. &

Phalen, D.N. Pathology of Pet and Aviary Birds (pp. 67-94). Ames: Iowa State Press.

Tell, L.A., Woods, L. & Cromie, R.L. (2001). Mycobacteriosis in birds. Revue scientifique et

technique (International Office of Epizootics), 20, 180-203.

Tortoli, E. (2003). Impact of genotypic studies on mycobacterial taxonomy: the new mycobacteria

of the 1990s. Clinical Microbiology Reviews, 16, 319-354.

Tortoli, E., Piersimoni, C., Bacosi, D., Bartoloni A., Betti, F., Bono, L., Burrini, C., De Sio, G.,

Lacchini, C. & Mantella, A. (1995). Isolation of the newly described species Mycobacterium

celatum from AIDS patients. Journal of Clinical Microbiology, 33, 137-140.

Valheim, M., Djønne, B., Heiene, R. & Caugant, D.A. (2001). Disseminated Mycobacterium

celatum (Type 3) infection in a domestic ferret (Mustela putorius furo). Veterinary Pathology,

38, 460-463.

Weisburg, W.G., Barns, S.M., Pelletier D.A. and Lane, D.J. (1991). 16S ribosomal DNA

amplification for phylogenetic study. Journal of Bacteriology, 173, 697-703.

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Figure legends:

Figure 1. Ventral view of the head of a white-tailed trogon. The skin is incised, exposing a

multilobulated bony mass which extends from the right hyobranchial apparatus.

Figure 2. Liver section showing multiple loosely arranged granulomas. A capsule of whirling

spindle shaped fibrocytes surround cental, relatively small, macrophages. HE, bar = 150 µm.

Figure 3. Pulmonary granuloma. Large number of multinucleated giant cells surrounding central

necrotic debris. HE, bar = 150 µm.

Figure 4. Severe granulomatous inflammation disrupting the cancellous bone of the ulna. Note the

irregular bony trabeculae and the necrotic focus (arrow). HE, bar = 800 µm.





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Figure 1. Ventral view of the head of a white-tailed trogon. The skin is incised, exposing a multilobulated bony mass which extends from the right hyobranchial apparatus.

80x59mm (300 x 300 DPI)

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Figure 2. Liver section showing multiple loosely arranged granulomas. A capsule of whirling spindle shaped fibrocytes surround a centre of relatively small macrophages. HE, bar = 150 µm.

80x80mm (300 x 300 DPI)

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Figure 3. Pulmonary granuloma. Large number of multinucleate giant cells surrounding central necrotic debris. HE, bar = 150 µm.

80x71mm (300 x 300 DPI)

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Figure 4. Severe granulomatous inflammation disrupting the cancellous bone of the ulna. Note the irregular bony trabeculae and the necrotic focus (arrow). HE, bar = 800 µm.

80x85mm (300 x 300 DPI)

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as for BW 80x59mm (300 x 300 DPI)

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Disseminated Mycobacterium celatum infection in a white-tailed trogon ( Trogon viridis )

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