Signalment:  

Two-year-old, hermaphrodite, veiled chameleon, (Chamaeleo calyptratus).Initially the chameleon exhibited open mouth breathing and declining appetite, eventually requiring forced feeding. The patient returned to the clinic 4 weeks later with severe dehydration and obvious weight loss. The chameleon was euthanized at that time.

Gross Description:  

The chameleon was in good nutritional condition with normal muscle mass and moderate coelomic adipose stores. A small amount of clear red-tinged fluid was present in the coelom. Diffuse red discoloration was evident in the expanded lungs and the liver was mottled tan and red with slight rounding of the margins. Ovotestes were present bilaterally. Mucoid content was evident in the lumen of the stomach and the colon; the intestinal content was otherwise scant.

Histopathologic Description:

Lung: The faveolar septa are expanded with moderate to marked congestion of the vasculature and myriad slender bacilli are present within the lumina of blood vessels; bacteria are both free within the lumina and present within the cytoplasm of macrophages. Hemorrhage, intravascular fibrin thrombi and necrotic cellular debris often accompany the bacterial colonies and scattered small aggregates of epithelioid macro-phages are present multifocally in the septa. In areas, there is necrosis of the faveolar epithelium and erythrocytes, proteinaceous fluid and cellular debris are noted with the faveolar spaces. The bacteria are acid-fast with the Ziehl-Neelsen stain. Similar intravascular bacteria and occasional small aggregates of macrophages containing bacteria are present in most organs (liver, pancreas, kidney, brain, spleen, skeletal muscle, adrenal glands, small intestine and ovotestes).

Morphologic Diagnosis:  

Necrotizing and histiocytic interstitial pneumonia, diffuse, subacute, with myriad intravascular/intrahistiocytic acid-fast bacilli and intravascular fibrin thrombi

Lab Results:  

Sections of lung were submitted for bacterial culture. Primary culture on a sheep blood agar plate after 48 hour aerobic incubation at 37^oC resulted in heavy growth of tiny smooth cream-colored colonies. Gram’s stain revealed pleomorphic gram-positive bacilli. The bacteria were acid-fast with the Ziehl-Neelsen stain. The isolate was sent to the National Reference Centre for Mycobacteriology (Public Health Agency of Canada). Based on 16s and hsp65 gene sequencing, the isolate had 100% sequence identity to Mycobacterium chelonae chemovar niacinogenes.

Condition:  

Pneumonia/Mycobacterium chelonae

Contributor Comment:  

Mycobacteria are ubiquitous in nature and can be isolated from the soil, dust, water and bioaerosols.¹   Reptiles are generally thought to acquire mycobacterial infections via ingestion or through defects / penetrating injury in the skin.¹¹ In this chameleon, there was a localized area of intestinal ulceration and granulomatous enteritis, suggesting that infection may have been acquired through the intestinal tract. In reptiles, like most species, mycobacterial infections tend to be chronic with rare acute infections reported.⁵ The typical gross lesions are grey-white nodules in multiple organs. Microscopically, early lesions are composed of organized collections of foamy macrophages that with time may become chronic granulomas composed of a mixture of epithelioid macrophages, lymphocytes, plasma cells, and multinucleated giant cells often surrounding a central region of necrosis and occasionally with a surrounding wall of fibrous connective tissue.¹¹ In this case, there were a few small early granulomas in multiple organs including the brain, lung, liver, kidneys and intestine. More striking, however, was the presence of myriad intravascular bacteria in multiple organs and frequent intravascular fibrin thrombi. These changes are compatible with acute to subacute infection with acute bacteremia and disseminated intravascular coagulation.

Mycobacteria are broadly divided into two groups: Mycobacterium tuberculosis complex and non-tuberculous myco-bacteria.² While only non-tuberculous mycobacteria have been reported to cause infections in reptiles, several different species have been associated with these infections. These include M. confluentis, M. chelonae, M. haemophilum, M. hiberniae, M. neoarum, M. confluentis, M. nonchromogenicum, M. marinum, and M. thamnopheos.^2,5,11  The most common causes of mycobacteriosis in reptiles are reported to be M. marinum, M. chelonae and M. thamnopheos.⁵  The isolate in this case was confirmed to be M. chelonae chemovar niacinogenes.

Non-tuberculous mycobacteria are classified into four Runyon groups according to growth rate and pigmentation.⁵ Runyon group I mycobacteria are slow growing and form pigment in the light following growth in the dark.  Runyon group II organisms are also slow growing bacteria; these bacteria form pigment in the dark following growth in the light. Runyon group III bacteria are slow growing and do not form pigment in the dark or light. Fast growing non-pigmented mycobacteria are placed in Runyon group IV.  These mycobacteria form mature colonies on solid agar within 7 days, while bacteria in Runyon groups I to III take longer periods of time for cultivation.²  Most mycobacteria which infect reptiles fall into Runyon groups I and IV.⁵  Mycobacterium chelonae is a rapidly growing mycobacteria belonging to Runyon group IV.⁴  Rapidly growing mycobacteria are relatively resistant to standard disinfectants and antibiotic treatment and are increasingly recognized as opportunistic pathogens in humans.^1,2

In reptiles, M. chelonei has been reported in association with osteoarthritis in a Kemp’s Ridley sea turtle,⁴ with stomatitis and subcutaneous granulomas in a boa constrictor,⁶ and with disseminated infection in a loggerhead sea turtle⁸ and a veiled chameleon.⁹ There is a single report of this bacterium causing acute fatal sepsis and disseminated intravascular coagulation in an eastern spiny softshell turtle⁸ with lesions very similar to those described in this case. Because of poor response to treatment and the zoonotic potential, euthanasia is often recommended for reptiles with myco-bacterial infections.

JPC Diagnosis:  

Lung: Pneumonia, histiocytic and necrotizing, multifocal to coalescing, moderate, with numerous intrahistiocytic and intravascular bacilli, veiled chameleon, Chamaeleo calyptratus.

Conference Comment:  

The contributor provides an outstanding review of non-tuberculous Mycobacteria in reptiles. Mycobacteria spp. are a large genus comprised of over 100 species of obligate pathogenic, potentially pathogenic, and environmental saprophytic bacteria.⁶ They are all morphologically similar and are composed of aerobic, gram-positive, acid-fast, non-spore forming bacilli.⁶ Conference participants were impressed by the large numbers of intrahistiocytic and intravascular thin filamentous bacilli that stain slightly basophilic on hematoxylin and eosin stained tissue section. These bacilli are intensely acid-fast positive with Fite-Faraco and Ziehl-Neelsen acid-fast stains, run by the Joint Pathology Center prior to the conference. Mycobacterium chelonae infection, confirmed by the contributor as the cause of rapid disseminated disease in this animal, is an opportunistic and potentially zoonotic pathogen that is characterized by rapid growth and high resistance to antibiotics.^1,2

As mentioned by the contributor, spontaneous non-tuberculous Mycobacteria sp., including M. avium, M. chelonae, M. szulagai, M. fortuitum, M. marinum, M. hemophilum, M. kansasii, and M. ulcerans have been reported to be the classic etiologic agents that cause histiocytic granulomas snakes, turtles, lizards, and crocodiles and should top the list of differential diagnoses for lesions similar to this case.^2,5,10,11 The obligate intracellular bacteria, Chlamydophila pneumoniae, can also occasionally infect reptilian species and induce histiocytic granulomas and should be considered as a differential diagnosis. Additionally, relatively recently described “Chlamydia-like” bacteria Parachlamydia acanthamoebae and Simikania negevensis, have been sporadically reported to form histiocytic granulomas in reptiles as well.¹¹

While histiocytic granulomas in reptiles are often induced by intracellular bacteria, such as in this case, heterophilic granulomas in reptiles are caused by extracellular pathogens, including most bacterial and fungal etiologies. Tissue injury can also induce heterophilic granulomas in reptiles.⁵ Heterophilic granulomas are characterized by accumulation and degranulation of heterophils leading to a central area of necrosis, stimulating a strong macrophage foreign body-like response. Caseocalcareous nodules, lymphoid infiltration, and peripheral fibrosis, typical of mammalian granulomas, have not been observed in reptilian heterophilic granulomas.⁵ Both histiocytic granulomas and heterophilic granulomas can progress to chronic granulomas, characterized by a fibrous connective tissue capsule, lymphocytes and plasma cell infiltration, and a central area of necrosis with a prominent lamellated appearance.⁵

References:

1. De Groote MA, Huitt G. Infections due to rapidly growing Mycobacteria. Clin Infect Dis. 2006; 42:1756–63.
2. Ebani, VV, Fratini F, Bertelloni F, et al. Isolation and identification of Mycobacteria from captive reptiles. Res Vet Sci. 2012; 93:1136–1138.
3. Fremont-Rahl JJ, Ek C, et al. Mycobacterium liflandii outbreak in a research colony of Xenopus (Silurana) tropicalis frogs. Vet Pathol. 2011; 48(8):856-867.
4. Greer LL, Strandberg JD, Whitaker BR. Mycobacterium chelonae osteoarthritis in a Kemp's Ridley Sea Turtle (Lepidochelys kempii). J Wildl Dis. 2003; 39(3):736-741.
5. Jacobson, ER. Bacterial diseases of reptiles. In: Jacobson ER, ed. Infectious Diseases and Pathology of Reptiles: Color Atlas and Text. Boca Raton, Florida: CRC Press; 2007: 461–526.
6. Mauldin E, Peters-Kennedy J. Integumentary system. In: Maxie MG, ed. Jubb, Kennedy, and Palmer’s Pathology of Domestic Animals. Vol 1. 6th ed. Philadelphia, PA:Elsevier; 2016:639-641.
7. Mitchell MA. Mycobacterial infections in reptiles. Vet Clin Exot Anim. 2012; 15(1):101–11.
8. Murray M, Waliszewski NT, Garner MM, et al. Sepsis and disseminated intravascular coagulation in an eastern spiny softshell turtle (Apalone spinifera spinifera) with acute mycobacteriosis. J Zoo Wildl Med. 2009; 40(3):572-575.
9. Nardini G, Florio D, DiGirolamo N, et al. Disseminated mycobacteriosis in a stranded Loggerhead Sea Turtle (Caretta caretta). J Zoo Wildl Med. 2014; 45(2): 357–360.
10. Reavill DR, Schmidt RE. Mycobacterial lesions in fish, amphibians, reptiles, rodents, lagomorphs, and ferrets with reference to animal models. Vet Clin Exot Anim. 2012; 15(1): 25–40.
11. Soldati G, Lu ZH, Vaughan L, et al. Detection of Mycobacteria and Chlamydiae in granulomatous inflammation of reptiles: A retrospective study. Vet Pathol. 2004; 41(4):388–397.

Click the slide to view.

---

3-1. Lung, chameleon:

3-2. Lung, chameleon:

3-3. Lung, chameleon:

3-4. Lung, chameleon:

---