Bovine pyogranulomatous mastitis caused by Mycobacterium goodii

Received 11 August 2014 Accepted 2 December 2014 Setor de Medicina Veterinária Preventiva Laboratório de Epidemiologia Veterinária (EPILAB), Universidade Federal do Rio Grande do Sul (UFRGS), 91540-000, Porto Alegre, RS, Brazil Departamento de Medicina Veterinária Preventiva Universidade Federal de Santa Maria (UFSM), Programa de Pós-Graduação em Medicina Veterinária, 97105-900, Santa Maria, RS, Brazil Centro de Biotecnologia Universidade Federal do Rio Grande do Sul (UFRGS), Programa de Pós-Graduação em Bioquı́mica, 91501-970, Porto Alegre, RS, Brazil Departamento de Patologia Veterinária Universidade Federal de Santa Maria (UFSM), Programa de Pós-Graduação em Medicina Veterinária, 97105-900, Santa Maria, RS, Brazil


Introduction
Mycobacteria are aerobic, non-motile, Gram-positive rods with a cell wall rich in mycolic acids and mycosides (Brown-Elliott & Wallace, 2002).Rapidly growing mycobacteria (RGM) are ubiquitous in the environment, can be isolated from soil and water, and need up to 7 days to grow on solid medium (Brown-Elliott & Wallace, 2002).RGM comprise the following groups: Mycobacterium chelonae-abscessus, Mycobacterium fortuitum and Mycobacterium smegmatis (Brown-Elliott & Wallace, 2002).Mycobacterium goodii was recently identified as a rapidly growing non-tuberculous mycobacterial species of the M. smegmatis group (Friedman & Sexton, 2001).This group may be divided with approximately 90 % accuracy based on their susceptibility to tobramycin (10 mg): M. smegmatis sensu stricto is susceptible to tobramycin (agar disk diffusion zone .30mm), M. goodii has intermediate susceptibility (agar disk diffusion zone 11-30 mm) and Mycobacterium wolinskyi is resistant (agar disk diffusion zone #11 mm) (Brown et al., 1999).
The diagnosis of RGM infections may be quite difficult by conventional methods (Lemarie, 1999).Although several phenotypic characteristics can help identify M. goodii, they do not have a strong discriminating power for Abbreviations: Abbreviation: RGM, rapidly growing mycobacteria.
The ecological niche of M. goodii remains unclear, although M. goodii has been acknowledged as a spotted hyena pathogen (van Helden et al., 2008) and has been found in dogs with a concurrent infection caused by hyperadrenocorticism (Bryden et al., 2004).Cases in large animals remain undiagnosed or misdiagnosed.Nontuberculosis mycobacterial bovine mastitis is uncommon, but it has been proved that the M. smegmatis group species may cause clinical mastitis in sheep and dairy cows (Thomson et al., 1988).Here, we describe an unusual case of bovine pyogranulomatous mastitis caused by M. goodii.

Case report
A 5-year-old Holstein cow belonging to a herd of 67 milking cows, originally located in the south of Brazil, showed mastitis signs of pronounced glandular hardening.The herd characteristics included a semi-intensive dairy system and the majority of the cows belonged to the Holstein breed.All animals were vaccinated against foot-and-mouth disease and brucellosis.Tuberculosis was routinely controlled, and veterinarians and technical staff of a cooperative provided assistance to the farmer.The cow underwent several intramammary (aminoglycoside and b-lactam) and parenteral (tetracyclines) antibiotic therapies with treatment protocols for 2 weeks.Due to the lack of adequate response, the owner requested milk bacteriological testing.
A biopsy sample of the affected mammary quarter was fixed in 10 % buffered formalin, paraffin embedded, sectioned at 4 mm and stained with haematoxylin and eosin and Ziehl-Neelsen/Fite (EasyPath) for acid-fast rods (Berg, 1953) in order to verify tissue damage.
The DNA from an M. goodii strain SB 314/96 isolate was extracted by lysis with cetyltrimethylammonium bromide (CTAB), according to Sambrook & Russell (2001).Molecular diagnosis was performed by analysis of the partial sequencing of triplicate DNA product of approximately 1500 bp amplified from the 16S rRNA gene using universal primers (Fredricks & Relman, 1998).The PCR product was sequenced with ACT Gene Ana ´lises Moleculares LTDA (Centro de Biotecnologia) using an automatic sequencer ABI-PRISM 3100 Genetic Analyzer (Applied Biosystems).The sequences were submitted to a consensus analysis based on chromatogram reliability obtained with the Staden Package Gap 4 program (Staden et al., 2000) and alignment by National Center for Biotechnology Information BLAST with DNASIS software (v.2.5; Hitachi Software Engineering Co.).In order to confirm the identification of the isolate as M. goodii, we performed a phylogenetic analysis based on the 16S rRNA gene using the neighbour-joining method.In order to calculate the evolutionary distances and the pairwise deletion of gaps, the p-distance was implemented with MEGA v.5.2.2 software.

Diagnosis
After 3 days of incubation, a slow and sparse growth of lightly pigmented, shiny and smooth colonies ranging from 1 to 2 mm was observed on the blood agar plate, and no growth was observed on the MacConkey plate.Bacterial growth was found in two of the eight milk samples collected (the same kind of growth was found in both samplings).Gram and Ziehl-Neelsen staining revealed poorly staining, irregular, slender Gram-positive and acidfast rods.Biochemical characterization of the isolate showed that it was catalase, urease, glucose, sucrose, mannitol and nitrate reduction positive and was negative for maltose, CAMP test, oxidase and aesculin.The isolate was considered to belong most probably to the M. smegmatis group due to its rapid growth, acid-fast staining and biochemical properties (Hartmans et al., 2006).In the antimicrobial susceptibility testing, the isolate was sensitive to all antimicrobials tested and was intermediate to tobramycin (agar disk diffusion 22 mm), showing the pattern for M. goodii.This in vitro susceptibility pattern is similar to previously reported findings (Brown-Elliott & Wallace, 2002).
Histologically, there were multiple foci of severe and diffuse inflammatory infiltration.They were characterized by a central area of necrosis with large numbers of neutrophils, mostly degenerated, surrounded by macrophages, epithelioid cells and Langhans giant cells, and fewer lymphocytes and plasma cells.Fibroblasts were observed in the peripheral zone.
The glandular ducts were filled with a large number of neutrophils and necrotic epithelioid cells.These microscopic changes are characteristic of pyogranulomatous and diffuse mastitis (Fig. 1), suggestive of bacterial intracellular infection.Histological slides stained with Ziehl-Neelsen showed poorly positive acid-fast rods (Fig. S1 available in the online Supplementary Material).Phenotypic characteristics can help identify M. goodii, although they do not have a strong discriminatory tool to differentiate M. goodii from other members of the M. smegmatis group.Thus, to assure the definitive identification of the isolate, we used a molecular approach.Here, although the 16S rRNA genes of M. goodii and M. smegmatis sensu strictu differed by only 3 bp, the phylogenetic analysis (Fig. 2) performed was a useful tool to differentiate these species.The consensus sequence showed 100 % nucleotide similarity with M. goodii sequences NR029341, Y12872 and AY457079 in GenBank.

Discussion
The various attempts at intramammary antibiotic treatment may have been the main reason for the introduction

Fig. 2 .
Fig. 2. Phylogenetic analysis based on 16S rRNA gene sequences showing the homology between the 16S rRNA gene sequence of M. goodii SB 314/96 (with a label) and other 16S rRNA gene sequences of Mycobacterium species.The neighbour-joining method using the p-distance to calculate the evolutionary distances and the pairwise deletion of gaps was implemented with MEGA v.5.2.2 software.The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to each branch.Corynebacterium jeikeium was used as the outgroup.