Mycobacteriosis Associated with Mycobacterium peregrinum Infection in Red-Crowned Cranes (Grus japonensis) in China

Mycobacteriosis of birds is a chronic progressive disease caused by Mycobacterium species, with Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium genavense being most commonly identified (Tell et al. 2001). Other mycobacterial species such as Mycobacterium fortuitum and Mycobacterium terrae have also been implicated as potential causes of mycobacterial infections in birds (Soler et al. 2009). Wild birds play an important role in the ecology and movement of mycobacterial species. Mycobacterium peregrinum, a fast-growing nontuberculous mycobacteria (NTM), belongs to the M. fortuitum group, and can cause mycobacteriosis in humans (Brown-Elliott and Wallace 2002) and birds (Vitali et al. 2006). We describe the identification of M. peregrinum as the cause of mycobacteriosis in Red-crowned Cranes (Grus japonensis). The Red-crowned Crane is a large East Asian crane and one of the most endangered crane species; only about 1,500 birds remain in the wild.

In May 2012, five adult Red-crowned Cranes aged 5–8 yr were found dead in Yancheng National Nature Reserve, Jiang Su, China. For several weeks previously, the cranes had exhibited signs of illness, including fever, moist rales, shortness of breath, and loss of appetite. Oral administration of erythromycin, amoxicillin, and cephalosporin incorporated into their feed was unsuccessful. Postmortem examination revealed white nodules of various sizes in the lungs and livers. A fast-growing Mycobacterium was isolated from these tissues after 7 days of incubation at 37 C under aerobic conditions. Colonies on modified Lowenstein-Jensen medium were yellow, dry, irregular, and rough. The bacteria were long, rod-shaped, gram-positive, and acid-fast, and adhered in an arrangement similar to branched hyphae. Spores, capsules, and true branching were not observed. Histopathologic examination revealed granulomatous inflammations with abundant macrophages and multinucleated giant cells.

Biochemical characterization of the strain was performed using the Vitek 2 identification system (bioMérieux, Marcy l'Étoile, France), which identified the isolates as Mycobacterium peregrinum (probability 99.3%). We tested the bactericidal activity of 13 antibiotics, alone and in two- and three-drug combinations, against M. peregrinum clinical isolates. An inoculum of approximately 10⁵ colony-forming units was cultured in 10 mL Mueller Hinton broth with individual drugs or drug combinations and incubated at 37 C for 4 days (Santos et al. 2008). Of the 13 antibiotics tested, moxifloxacin showed the highest bactericidal activity against M. peregrinum, either alone or in combination with other antibiotics tested (Table 1).

To further characterize the isolates, molecular identification using 16S rRNA, rpoB, and hsp65 genes were performed (Aranaz et al. 2008). The isolates were confirmed as belonging to the genus Mycobacterium by PCR amplification and sequencing of the 16S rRNA and rpoB genes. These PCR assays target a 1,030-bp sequence specific to 16S rRNA of Mycobacterium spp. and a 136-bp sequence of the rpoB gene (Table 2), which can discriminate between the Mycobacterium tuberculosis complex and NTM (Kim et al. 2004). Identification of isolates to species level was carried out by PCR amplification and sequencing of the 16S rRNA and the 65-kDa heat-shock protein (hsp) genes (Table 2). The isolates were designated as JS-201205, according to their origin, and the 16S rRNA sequence was deposited in GenBank (accession KC292269). According to a Basic Local Alignment Search Tool search (NCBI 2013), strain JS-201205 was 100% similar to previously deposited sequences of M. peregrinum (accessions JX266704, HE575962, and AM884581). The strain was further confirmed as M. peregrinum according to the 441-bp product by hsp65, which was used for the identification of fast-growing mycobacteria to species level (Aranaz et al. 2008).

Sequence alignment was performed using the Clustal W multiple alignment in the MegAlign program of DNASTAR (DNASTA, Inc., Madison, Wisconsin, USA). Phylogenetic analyses were performed based on the 16S rRNA and hsp65 genes by the neighbor-joining method as implemented in MEGA 5.2 (Tamura et al. 2011). Phylogenetic analysis of existing M. peregrinum isolates revealed that strain JS-201205 clustered with other M. peregrinum isolates, with a 98% bootstrap value with 16S rRNA and 87% with hsp65 (Fig. 1).

Red-crowned Cranes were kept in semicaptivity in the nature winter habitat reserve and allowed to come and go freely. How Red-crowned Cranes become infected with M. peregrinum is unclear, but the bacteria are free-living in water and soil, so waterborne transmission among cranes seems likely.

Mycobacterium peregrinum can also cause infections in humans. These infections often involve the skin, are difficult to treat, and require long-term antibiotic therapy. Mycobacterial infections in humans are associated with exposure to fish or contaminated water (Aranaz et al. 2008). Over the last decade, a small but increasing number of sporadic human infections with M. peregrinum have been reported (Ishii et al. 1998; Rodríguez-Gancedo et al. 2001; Short et al. 2005). Although the transmission of M. peregrinum to humans through exposure to contaminated water is very likely, the potential for accidental infections of breeders from handling infected cranes is of concern.

We have demonstrated that M. peregrinum causes mycobacteriosis in wild birds. This infection could have important implications for the conservation of endangered species, and possible public health risks should be considered.

We acknowledge the Wildlife-borne Diseases Surveillance Project from State Forest Administration, the joint project of the US Department of Agriculture and the Institute of Zoology-Chinese Academy of Sciences, National Natural Science Foundation of China (31072126, 31101806), and Beijing Poultry Industrial Technology System and National Science and Technology Pillar Program during the Twelfth Five-Year Plan Period (2013BAD12B04).