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Abstract
A disseminated Mycobacterium avium subsp. avium infection was diagnosed in a pet Korean squirrel. Grossly, multiple small nodules in the lung, liver, spleen, and skin were observed. Adrenal glands were very enlarged. The only tissue exhibiting necrosis and calcification was a very enlarged bronchial lymph node. The remaining lymph nodes were slightly enlarged. Moderate ascites was also observed. Microscopically, a disseminated granulomatous inflammation with numerous lymphocytes was seen. Acid-fast bacilli were detected in macrophages, in giant cells, free in the interstitium, and in some lymphatic vessels, both within cells and free in the lumen. M. avium subsp. avium was isolated and identified by polymerase chain reaction–restriction endonuclease analysis.
Key words: Granulomatous inflammation; Mycobacterium avium subsp. avium; PCR; pets; restriction endonuclease; squirrels; zoonosis.
Exotic animals are becoming increasingly popular as pets. Although their health status is generally well controlled, the risk of transferring zoonotic pathogens from infected pets should not be overlooked. Squirrels are one of these popular pets, and although studies on squirrel diseases are scarce and mainly refer to wild individuals,3,13,15 some zoonotic diseases have been described.3 Diseases such as toxoplasmosis, campylobacteriosis, yersiniosis, leptospirosis, typhus, or the infection by some aggressive species of the bacterium Staphylococcus, as well as some parasitic infections, are the most frequently reported.3,13–16
Mycobacterium avium subsp. avium (Maa) is the most common cause of disseminated bacterial infection among human acquired immunodeficiency syndrome (AIDS) virus–infected individuals in developed countries, both in the USA and Europe.2,7 Infection typically occurs late in the course of AIDS when a clear immunodepression occurs, with systemic spread being a common feature of the disease. Maa infection has usually been described in birds and, more sporadically, in farm, pet, and exotic animals.17 Some studies have revealed that Maa strains isolated from human beings are identical to pig isolates and different from bird isolates.11 To our knowledge, however, it has not been reported in squirrels, and mammalian tuberculosis caused by the complex bovis–tuberculosis should be included as differential diagnosis of granulomatous diseases in these species. The other species included in the avium complex are M. avium subsp. silvaticum (Mas) and M. avium subsp. paratuberculosis (Map). Map is the cause of a granulomatous enteritis of ruminants called paratuberculosis or Johnés disease, and Mas can be involved in tuberculosis in birds and paratuberculosis in mammals.18
A 4-year-old male Korean squirrel was referred to the Diagnostic Service of Neiker (Basque Institute for Agrarian Research and Development) for postmortem examination. The squirrel had died suddenly with only some strange movements just before death. The squirrel had been bought in a pet shop 4 years ago and fed chestnuts, apples, bananas, and cherries. The squirrel had not suffered any disease during his life, and no symptoms were observed in the days before its death. The owner had previously another squirrel that died in a similar way. The death was thought to be related to food, but no postmortem examination was performed on the first squirrel.
After postmortem examination, samples for histopathologic, microbiologic, and polymerase chain reaction (PCR) analysis were taken. For histopathologic studies, tissues were fixed in 10% neutral buffered formalin, embedded in paraffin wax, cut at 4 µm, and stained with HE and Ziehl–Neelsen method for acid-fast bacteria. Peritoneal fluid was submitted to direct Ziehl–Neelsen staining and culture as described previously.1 Briefly, 1 ml of the fluid was homogenized with 20 ml of 0.75% hexadecilpiridinium solution and left for 18 hours. Then the upper part of the sediment was collected with a plastic disposable pipette, and 4 tubes of Coletsos medium (bio-Mérieux sa, Marcy 
Etoile, France) were inoculated with 3 drops each. The tubes were incubated at 37°C and inspected weekly with the aid of a stereoscopic microscope for the presence of any growth. Molecular methods were used to identify the colonies obtained. Two different PCR tests specific for the M. bovis–M. tuberculosis complex and M. avium subsp. silvaticum, respectively, were performed on the colonies according to previously published procedures.6,12 A PCR–restriction endonuclease analysis on the basis of polymorphisms in insertion sequence IS1311 as described by Marsh et al.10 was carried out to discriminate between Maa and Map.
At necropsy, several white small nodular lesions, 1–2 mm in diameter, were mainly observed in the lung and less clearly in the liver and the spleen. They were randomly distributed throughout the parenchyma. A similar lesion was seen in the skin, located in the ventral thoracic area close to the sternum. No necrosis or calcifications were appreciated in the sections of these nodules, except in the bronchial lymph node, which appeared very enlarged and showed a large necrotic mass, about 8–10 mm in diameter, occupying all the parenchyma. The other lymph nodes were slightly enlarged, whereas the liver and the spleen were moderately enlarged and the adrenal glands very enlarged. A moderate amount of ascitic liquid was observed.
Microscopically, a granulomatous inflammatory reaction was observed in the lung, liver, spleen, kidney, adrenal gland, pancreas, peritoneum, skin, and lymph nodes. The intensity of the infiltrate was most severe in the lung, liver, spleen, adrenals, and pancreas; moderate in the skin; and minimal in the kidney and heart. This infiltrate consisted of macrophages, giant cells, and numerous lymphocytes. In the lung, macrophages predominated, forming granulomas surrounded by lymphocytes. Scattered giant cells, sometimes forming granulomas, were also observed (Fig. 1). A moderate to marked lymphoid hyperplasia around blood vessels and airways was also found. In some areas, a slight neutrophilic alveolitis and bronchiolitis was also visible. The bronchial lymph node showed a large central necrotic area surrounded by numerous giant cells, macrophages and lymphocytes, and collagen fibers. A great number of these giant cells showed bizarre shapes varying from round to irregular and often had numerous nuclei. Some of them harbored as many as 140 nuclei (Fig. 2). In some areas of the inflammatory capsule, scattered small necrotic foci were observed. No necrosis or bizarre giant cells were found in other organs. Calcification was very scarce and only observed in a small area of the necrotic mass in the bronchial lymphoid node. In the liver, spleen, and pancreas, however, a lymphocytic infiltrate with scattered small granulomas was the common presentation. In the liver, a moderate and diffuse lymphocytic perivasculitis was also observed. In the adrenal glands and the skin, a mixed infiltrate of macrophages, giant cells, and lymphocytes was detected. In the adrenal glands, macrophages and giant cells were predominantly observed in the medulla, whereas lymphocytes were mainly confined to the cortex. In the kidney and heart, a mild localized inflammation, mainly composed of lymphocytes, was observed. Acid-fast bacilli were extremely abundant in the bronchial lymph node, moderately abundant in the adrenal glands, and scattered in the other organs. In the lymph node, they were observed within giant cells and macrophages as well as free in the interstitium or in some lymphatic vessels, both within monocytes and free in the lumen. In other organs, they were only observed in macrophages and giant cells.
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Maa shows a widespread environmental distribution, the main sources being water and soil. It is typically isolated from birds, although some cases in mammals are also reported.17 This squirrel was kept as a pet for a period of 4 years and had no contact with other animals, except a short contact with a pet bird a few days 1 year previous to its death. Because the owners had another squirrel before this one that died in a similar way and because mycobacteria are very resistant in the environment, it might be possible that the previous squirrel died from a Maa infection as well. Because no postmortem examination was done, this remains speculative. In avian species, lesions are typically found in the liver, spleen, and intestines, with lung involvement being uncommon. Localization depends on several factors, such as avian species, mycobacterial species, immune status of the bird, and feeding or behavioral habits. In pulmonary cases, an airborne source of infection, such as dust or aerosols, has been suggested. Taking into account that this squirrel showed the most extensive lesions in the respiratory system, an aerogenous exposure was the likely route of infection.17
Because of the nonprogressive and chronic character of this infection, generalized lesions are usually uncommon, although some cases of disseminated disease have been described in zoo ruminants and immunosuppressed dogs and cats.5,8,17 Under natural conditions, however, extensive lesions by Maa in mammals, other than rabbits and swine, as seen in poultry are very exceptional. In this case, a generalized infection involving several organs, including lung, liver, spleen, pancreas, and skin, was observed. Although it is impossible to be sure that the squirrel was not immunocompromised, because of its age and the absence of previous recurrent disease, the authors assumed that the infection developed in an immunocompetent squirrel. This would also be supported by the ability of Maa to cause a generalized infection in rodents, of which squirrels are a species.4
In general, the inflammatory reaction showed a marked lymphoid infiltration. Macrophages and giant cells only predominated in the respiratory system, especially in the bronchial lymph node. Except for bronchial lymph node, necrosis was not observed, and very scarce mineralization was associated with this lesion. Mycobacteria were mainly associated with the necrotic lesions and macrophages and giant cells. Lesion features and bacillus location are similar to those described for the avian type tubercle bacillus infection in rodents and are correlated to the immune response of the host.4
Maa is an opportunistic pathogen in immunodepressed human beings. It usually causes a disseminated infection in late AIDS patients when a typical CD4 T cell decrease is found.9 The squirrel was kept in close contact with owners and probably shed mycobacteria, resulting in a contamination of the environment with Maa. Thus, transmission to owners might have occurred, but no history of disease was found in them.
To our knowledge, this is the first report of a disseminated M. avium subsp. avium infection in a pet squirrel. Mycobacterial infections should be considered zoonotic infections in squirrels. Although the infection seems to be sporadic, taking into account that Maa infection can occur in immunocompromised humans, care should be taken by owners and people manipulating pet squirrels.
References
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