Coinfection by Mycobacterium marinum and Mycolicibacterium fortuitum in a captive adult diamondback water snake causing disseminated mycobacteriosis with acute cutaneous ulceration

Abstract

An adult male captive diamondback water snake (Nerodia rhombifer) was found dead after a 1-d history of lethargy and cutaneous ulcers. The snake had eaten 2 sunfish (Mola spp.) 5 d before death. Gross examination revealed white-to-tan nodules in the lung and liver and segmental intestinal impactions with digested fish. Histopathology confirmed disseminated granulomas with numerous intrahistiocytic acid-fast bacteria in the skin, skeletal muscle, lung, liver, and intestines. Mycobacterium marinum and Mycolicibacterium fortuitum were identified by culture of the hepatic granuloma, followed by PCR and rpoB gene sequencing. To our knowledge, this is the first description of M. marinum and M. fortuitum coinfection in this species. Although M. fortuitum has been isolated from reptiles, lesions associated with its presence in tissues have not been described previously. Interestingly, the mineralization within granulomas that we observed in our case is not reported in mycobacterial infection in reptiles, whereas this finding is common in mammals.

An adult male captive diamondback water snake (Nerodia rhombifer) from the Greater Baton Rouge Zoo was presented for autopsy at the Louisiana Animal Disease Diagnostic Laboratory (LADDL; Baton Rouge, LA, USA) on 2017.02.15. The animal was captured from the wild as an adult in October 2014, and no prior history of disease in captivity had been documented. Five days before death (2017.02.10), the snake predated and ate 2 sunfish (Mola spp.). The red cutaneous lesions appeared on 2017.02.14. The animal was found dead following a 1-d history of lethargy.

On postmortem examination, the snake weighed 246 g and was 87 cm long. Significant gross findings included cutaneous ulcers and necrotic lesions, which extended to the subcutis, throughout the body including the right ventral cervical region, the left dorsal aspect of the proximal body, the dorsal and left ventral midbody, and the left caudal region (Fig. 1A). The gingival mucosa of the right mandible had a focally extensive area of red discoloration. The right lung and liver had multifocal-to-coalescing, slightly raised, white-to-pale-tan nodules of pinpoint to 0.5-cm diameter that extended into the parenchyma (Fig. 1B). The gastric serosal surface had dark-brown-to-gray, friable, 0.2-cm nodules that extended into the gastric wall. The gastric mucosa was red in multifocal-to-coalescing areas. The small and large intestines had numerous distended segments, and the intestinal wall was transmurally red-to-dark-red (Fig. 1B). The distended intestinal segments were filled with dense, dry, gray-to-light-brown digesta mixed with remnants of fish bones. In the lumen of the small intestine of non-affected segments, five 1.5–3.5-cm long tapeworms were present. A 3.5 × 0.1 × 0.1-cm white segmented worm was embedded in the body wall muscles near the cranial third of the right lung, with partial protrusion of the tail side into the coelomic cavity. One gray-to-pale-tan ~0.7-cm long C-shaped worm was encysted in the mesentery.

Figure 1.

Disseminated mycobacteriosis in a diamondback water snake (Nerodia rhombifer). A. Multifocal cutaneous ulceration and necrosis (arrowheads). Inset: cutaneous lesions extended to the subcutis, throughout the body including the right ventral cervical region (arrowhead). B. Slightly raised, white-to-pale-tan, pinpoint-to-0.5-cm nodules extended into the hepatic parenchyma (arrowheads). The small and large intestines had distended segments, and the intestinal wall was transmurally red-to-dark-red (asterisk). C. The hepatic parenchyma is effaced by granulomas composed of a central area of necrosis and degenerate leukocytes with occasional mineralization (asterisks) surrounded by epithelioid macrophages and multinucleate giant cells (Langhans and foreign-body types) admixed with scattered heterophils. H&E. Bar = 250 μm. D. Myriad intrahistiocytic acid-fast–positive bacilli. Ziehl–Neelsen stain. Bar = 20 μm.

On histologic examination, ~80% of the normal hepatic parenchyma was replaced by granulomas composed of a central area of necrosis and degenerate leukocytes with scattered mineralization surrounded by numerous epithelioid macrophages and multinucleate giant cells (Langhans and foreign-body types) admixed with scattered heterophils (Fig. 1C). Mild focal infiltrates of lymphocytes and rare plasma cells were at the periphery of the granulomas. Numerous intrahistiocytic acid-fast–positive bacilli were highlighted with Ziehl–Neelsen (ZN) stain (Fig. 1D). Granulomas containing intrahistiocytic acid-fast–positive bacilli were obs-erved in histologic lesions in the lung, the skin and underlying skeletal muscle, segments of intestine, and mesentery.

In the cutaneous lesions, granulomas containing acid-fast organisms effaced the dermis and muscularis; the epidermis was markedly necrotic, ulcerated, and replaced by abundant serocellular crust, hemorrhage, fibrin, edema, and numerous superficial basophilic bacterial colonies. Fungal hyphae with thin variably parallel walls, non-pigmented, and infrequent distinct septations (Gridley stain positive) were observed in some of the lesions. Within the lesser affected epidermis, intercellular edema was frequently present.

In the red distended intestinal segments, abundant granulomas effaced the intestinal walls transmurally and contained acid-fast bacteria. Additionally, the submucosa of the intestine was expanded by foci of amorphous hyalinized eosinophilic material, mixed with colonies of coccoid bacteria, frequent mineralization rimmed by epithelioid macrophages, and moderate numbers of heterophils (degenerate parasites, presumptively). Occasionally, the lamina propria was markedly expanded by parasites consistent with cestodes with or without mineralization and surrounded by histiocytic inflammation and a fibrous capsule.

In the oral cavity, there was an extensive ulcerated area. The exposed submucosa was infiltrated by variable numbers of heterophils, which extended into the deeper tissue around teeth. Exudate in the lumen was composed of heterophils, fibrin, cellular debris, and proteinaceous material. Significant lesions were not evident in the other tissues.

Aerobic culture performed in samples from liver, lung, oral, and subcutaneous swabs yielded an Aeromonas spp. growth. Aerobic culture on skin swabs, and mycobacterial and Salmonella cultures performed on a pool of liver and lung samples did not yield bacterial growth. Mycobacterium marinum and Mycolicibacterium fortuitum (formerly Mycobacterium fortuitum) were identified by culture of the hepatic granuloma, followed by PCR and rpoB gene sequencing, yielding a final diagnosis of mycobacteriosis. The other bacteria noted histologically were secondary opportunistic invaders. Sheather flotation tests revealed Eimeria spp. The parasite found in the body wall was identified as a pentastomid (Porocephalus crotali) based on the anatomic localization, host, and the 4 hooks surrounding the key-shaped mouth on the cephalothorax. ²⁴ The encysted parasite from the mesentery was identified as a Spirometra mansonoides plerocercoid. Fragments of parasites found in the small intestines were unable to be identified given marked autolysis; however, the parasites were presumed to be S. mansonoides plerocercoids in view of the encysted plerocercoid of this parasite within the mesentery. Finally, no fungi were retrieved from the mycology culture performed on a pool of liver and lung tissue. Fungal culture of the skin was not performed.

In reptiles (turtles, crocodiles, lizards, snakes), mycobacteriosis is a sporadic disease. ⁴ Most of the agents involved are ubiquitous in the environment (soil, dust, water, plants) and the prevalence of the infection is relatively low.^4,5,13,17,20 Cold-blooded animals are considered naturally resistant to mycobacteria because they carry the organisms subclinically. ⁵ Typically, only predisposed animals become diseased. ¹³ Animals contract the infection through lesions in the integumentary, respiratory, or urogenital systems, and by ingestion of contaminated food (particularly fish or amphibians) or water.^5,10,17,20 Immunodeficient poikilotherms are at risk.^4,10,17,20 Other predisposing factors include stress, inadequate nutrition, and comorbidities. ⁵ Clinical signs are related to the location of lesions.^5,20 We retrieved no cases of mycobacterial infection caused by M. marinum and M. fortuitum in a diamondback water snake with the uncharacteristic mineralization of the granulomas in a comprehensive search of Google, PubMed, CAB Direct, Web of Science, and Scopus, using search terms: Nerodia rhombifer mycobacteriosis; diamond back water snake mycobacteriosis; Mycobacterium marinum and Mycobacterium fortuitum coinfection; and snake Mycobacterium marinum and Mycobacterium fortuitum infection.

Mycobacterial infections are reported in captive snakes, but not as commonly as in other reptiles. Most of the reported cases have oral and pulmonary lesions.^9,17 Most nontuberculous mycobacteria can induce bacteremia and reach several internal organs. ⁵ The distribution of the lesions varies from localized (oral, pulmonary, oviductal, or dermal) to disseminated (lung, liver, kidney, heart, bone, gonads, nervous system, joints).^4,5,10,17

Mycobacterial infections typically induce the formation of granulomas in reptiles.^4,10 Older granulomas may be surrounded by fibrous connective tissue.^4,5,12,13,17 Unlike mammalian tubercles, mineralization has not been reported in reptiles.^{3,11,14,18,21,22,25} To our knowledge, dystrophic mineralization associated with mycobacterial infection has not been reported previously in reptiles. Acid-fast histochemical stains can demonstrate bacteria within granulomas; Fite–Faraco staining can be more sensitive than ZN staining.^12,13,20 However in our case, the ZN stain revealed the abundant organisms within the hepatic granulomas.

Disseminated granulomas have been reported in poikilothermic animals, infected for instance by Mycobacterium chelonae, M. confluentis, M. fortuitum, M. haemophilum, M. hiberniae, M. intracellulare, M. kansasii, M. kumamotonense, M. marinum, M. neoaurum, M. normochromogenicum, M. phlei, M. smegmatis, and M. ulcerans.^4,5,10 M. marinum is the most common isolate from reptiles, followed by M. chelonae and M. thamnopheos.^8,10,17 Nontuberculous mycobacterial species reported in snakes include M. chelonae, M. fortuitum, M. genavense, M. haemophilum, M. kansasii, M. leprae, and M. marinum.^4,12,22,25

M. marinum can infect reptiles kept in captivity. Several reptile species, including crocodiles, turtles, lizards, snakes, and amphibians (salamanders, toads, frogs) are susceptible to M. marinum infection and the development of systemic lesions. ² Environmental temperature is a relevant physical property determining the rate of infection in reptiles alongside other predisposing factors, including stress, inadequate nutrition, and comorbidities.^2,5 Poikilothermic animals, inoculated intraperitoneally with M. marinum, develop an infection at the optimal temperature of 30°C. ² M. marinum infection is observed in people who have contact with reptiles, fish tanks, aquaria, fish, and other aquatic animals, and causes the condition termed “fish tank granuloma.”^1,14,15 Several reports suggest that poikilothermic animals such as fish can be a source of infection.^1,2,5,17

The most common source of exposure of reptiles to mycobacteria is thought to be contaminated water and infected food. ¹⁰ In our case, the snake ate a presumably infected Mola fish present within the snake’s enclosure at the zoo, 5 d prior to death. We speculate that the infected food was the source of infection in this case. Alternatively, the snake could have been a subclinical carrier of mycobacteria and succumbed to disease after a stressful event. However, we do not favor this hypothesis. The snake had been in captivity since 2014, and there was no record of previous health issues. Our findings contradict the paradigm of natural mycobacterial infections in reptiles, which are usually reported as chronic, progressive, and debilitating diseases.^6,9,13 However, some cases are reported to have an acute presentation, with systemic dissemination and cutaneous lesions. ¹³ In the absence of cutaneous lesions, clinical signs are nonspecific and includes anorexia and chronic weight loss. ¹³ In our case, we conclude that systemic mycobacteriosis, with acute cutaneous involvement was present within 5 d of ingestion of the infected fish.

Experimental intraperitoneal inoculation of M. marinum in a Thamnopis sirtalis sirtalis (eastern garter snake) led to the development of visceral granulomas and death of the animal as soon as 2–4 d post-inoculation. ² In zebra fish, some strains of M. marinum caused hemorrhages and inflammation within 5–16 d post-infection, which denotes rapid growth. ²³ These experimental studies support the rapid dissemination of M. marinum in animals, as in our case. Reported cases of disseminated mycobacteriosis in snakes, with skin or subcutaneous involvement, are limited to one case of a Boa constrictor infected with M. thamnopheos, with cutaneous, oral, and pulmonary lesions. ⁸ A second case of a B. constrictor, infected with M. chelonei, had evidence of subcutaneous granulomas on the dorsum and stomatitis. ¹⁶ Cases of cutaneous mycobacteriosis in reptiles that progressed to systemic infections have been documented in the literature. ¹⁹ Thus, possible systemic involvement secondary to a primary cutaneous mycobacterial infection cannot be ruled out completely in our case.

M. fortuitum is highly prevalent in the aquatic environment. ²⁶ Although M. fortuitum have been isolated from several species of reptiles, including lizards, turtles, and pythons, clinical cases associated with this agent in reptiles have not been reported in the literature. ⁴ Unlike the reptiles, M. fortuitum has been isolated from fish with or without clinical lesions. ²⁶ This is the first evidence of M. fortuitum associated with clinical disease within this class, to our knowledge.

Conventionally, histologic evaluation allows the diagnosis of mycobacteriosis by demonstration of acid-fast bacteria within the lesions.^17,20 Nevertheless, this method cannot provide the species.^17,20 The rpoB gene, the RNA polymerase β-subunit, is a well-characterized target on bacteria. ⁷ The use of rpoB gene sequencing is a useful technique to identify Mycobacterium isolates in veterinary medicine, to the clade and species levels, especially when 16S rRNA sequencing alone is not adequate to determine specific taxa.^7,17,20

Given that mycobacterial infection causes granulomatous inflammation, it must be considered as a differential diagnosis in any case of granulomatous disease in reptiles; however, not all granulomas in reptiles are caused by mycobacteria.^10,20 In fact, the reported and estimated rate of reptilian mycobacteriosis with granulomatous inflammation is 15.6–25.6%, ²¹ which highlights the need for ancillary testing to reach a definitive diagnosis. Coinfection by M. marinum and M. fortuitum was not documented previously in the literature. There is one report of coinfection by M. marinum and M. haemophilum in a royal python (Python regius). ⁶