Corynebacterium pseudotuberculosis is an intracellular bacteria and the etiologic agent of caseous lymphadenitis in domestic and wildlife species. We report C. pseudotuberculosis infection in Patagonian huemul (Hippocamelus bisulcus) from the Cerro Castillo National Reserve, Region of Aysen, Chile. Subcutaneous abscesses in the abdominal and pectoral regions from two animals were sampled and bacteriologic isolation was performed. In both cases, we isolated a C. pseudotuberculosis strain belonging to the ovine genotype. In addition, one isolate was resistant to ciprofloxacin and streptomycin. We report that H. bisulcus is a susceptible species to this bacterium, which is transmitted by direct or indirect contact with domestic sheep (Ovis aries) and which represents a potential conservation threat to populations of H. bisulcus. Additional research and prevention efforts should be addressed.
Corynebacterium pseudotuberculosis is a cosmopolitan agent that causes caseous lymphadenitis (CLA) disease in domestic sheep (Ovis aries) and goats (Capra aegagrus hircus). Additionally, the bacterium produces abscesses in horses (Equus caballus), ulcerative cutaneous granulomas in cattle (Bos taurus) (Baird and Fontaine 2007), and lesions in other domestic and wildlife species (Tarello and Theneyan 2008; Colom-Cadena et al. 2014) including cervids from North America and Europe (Kelly et al. 2012; Matos et al. 2015). In Chile, CLA is an endemic disease and could generate important economic losses in domestic animals (Tadich et al. 2005; Baird and Fontaine 2007), although ecologic or health effects in wildlife are unknown.
The Patagonian huemul (Hippocamelus bisulcus) is an endemic, midsized deer of the southern Andes of Chile and Argentina found in stepped and irregular foothills at elevations up to 1,300 m in the Andes Mountains. The huemul is considered an endangered species with fragmented populations and declining numbers, listed in the IUCN Red List of Threatened Species (2016). Although no sound population figures exist, it is estimated that there may be 2,000 individuals (Vila et al. 2006). Here we report the detection of a C. pseudotuberculosis strain from Patagonian huemul in the Region of Aysen, Chile.
During October 2015, a free-ranging, adult, male huemul living in the Cerro Castillo National Reserve (46°01′58′′S, 71°58′37′′W), Chilean Patagonia, was observed with a lateral subcutaneous abscess in the abdominal body region (Case 1). Its capture was performed under supervision and following recommendations of the Chilean authority. Once anesthetized with medetomidine (0.09 mg/kg) and ketamine (2 mg/kg), samples were obtained for antibiotic testing. The abscess was drained and then cleaned with sterile NaCl 0.9% solution. The animal was in a healthy condition without any other signs of disease. As a precaution, it was transported in a cage to a closed area within the park and maintained under veterinary care until healing. After 2 wk, the animal was released into the same Reserve. Later, during February 2016, a second healthy, free-ranging, adult male was observed within the same Reserve, 8 km away from the first case (45°58′46′′S, 71°56′01′′W), with a subcutaneous abscess in the left side of its pectoral area (Case 2). The capture and sampling of the abscess was performed as mentioned for Case 1. However, this animal was released in the same place immediately after recovery from anesthesia.
Because coinfection with other biologic agents has been reported (Muller et al. 2011; Matos et al. 2015), several diagnostic procedures were performed with the samples, although unique findings were related to the C. pseudotuberculosis infection (see Supplementary Table). Skin biopsies showed many neutrophils with abundant necrotic debris and Gram-positive bacilli. Abscesses contained a soft and pasty exudate resembling CLA lesions observed in goats.
For bacterial isolation, abscesses were sampled using sterile swabs and Cary-Blair transport medium (Copan Transystem®, Brescia, Italy). At the lab, swabs were seeded onto trypticase soy agar (Difco®, Sparks, Maryland, USA), plates supplemented with 5% (v/v) sheep blood, and then incubated at 37 C for 48 h. Suspicious Gram-positive bacteria were confirmed by biochemical characterization (API Coryne, Biomerieux®, Lyon, France). The cyclic AMP (cAMP) and cAMP-inhibition tests and the PCR assay for phospholipase D gene detection were made following protocols previously reported (Dorella et al. 2006; Pacheco et al. 2007; Retamal et al. 2011). Amplification and purification of the hypervariable rpoB gene segment was performed as previously described (Retamal et al. 2011), and automated sequencing was developed at the Faculty of Sciences, University of Chile, Santiago. The disk diffusion method of the Clinical and Laboratory Standards Institute criteria (2013) was followed to determine antimicrobial susceptibility (Table 1).
These assays confirmed the detection of the C. pseudotuberculosis bacterium in abscesses of H. bisulcus. The sequencing of the rpoB hypervariable segment (GenBank accession no. KY560450) revealed a complete nucleotide identity to the ovine genotype (GenBank accession no. HQ401568), and phenotypic resistance for the antimicrobials ciprofloxacin and streptomycin was detected in one isolate (Table 1). These are drugs that are frequently used in domestic animals in Chile.
The presence of C. pseudotuberculosis has been detected in slaughtered sheep from the Region of Aysen, with the highest detection rate (11.8%) in adults (Tadich et al. 2005). The risk for infection of wildlife inhabiting the Cerro Castillo National Reserve is probably the herds of domestic sheep located in the vicinity (within 5 km) of its borders. There are 69 herds with around 6,300 animals, most of which (70%) are ewes.
Microbiologic and epidemiologic evidence support the conclusion that sheep are the most probable source of C. pseudotuberculosis infection for free-ranging deer living in the Reserve. This contact between infected sheep and huemul could have occurred indirectly through pus-contaminated fomites, in which bacteria can survive for weeks or months (Dorella et al. 2006; Baird and Fontaine 2007). The rupture of contaminated abscesses releases a high number of bacteria which contaminate the environment and particularly the plants of the deciduous forest where these animals feed. Once infection was established in some individual huemul, transmission and dissemination of bacteria in this population may also have occurred by direct contact. Nevertheless, other predisposing factors could also be at work, as the geographic relationship between the wild and domestic species started almost 100 yr ago, yet the disease had not been previously reported for H. bisulcus. On the other hand, the natural course of untreated animals in the wild is unknown.
Whatever the transmission route, the exposure to a pathogen from domestic animals occupying their natural habitat is a real threat for H. bisulcus which, in addition to poaching and attacks from feral dogs (Vila et al. 2006; Briceño et al. 2013), constitutes a conservation challenge that should be addressed in the future by competent institutions and researchers. During 2015 and 2016, 40 huemul specimens were seen along 27 km of the Carretera Austral (CH-7) highway crossing the Reserve, 13 (33%) of which had CLA-compatible lesions. Because of the results from our work and from the visual observations, current efforts are being developed at a population level for a better characterization of the disease in Patagonian huemul from the Cerro Castillo National Reserve. Every new abscess in a living or dead specimen should be analyzed at the laboratory to determine the presence of the bacterium, its genotypic and phenotypic traits, and the pathologic consequences for animals.
We thank Consuelo Foerster (Zoológico Nacional), Mario Alegría Cea (Corporación Nacional Forestal), José Cerna Abarca (Programa de Protección al Huemul), and laboratory personnel from the Region of Aysen for their collaboration during this study. We also thank Cristobal Briceño for a critical review of the manuscript. This work was funded by Servicio Agrícola y Ganadero, Corporación Nacional Forestal, and the Universidad de Chile.
Supplementary material for this article is online at http://dx.doi.org/10.7589/2016-09-213.