First report of Mycobacterium avium subsp. hominissuis in a black howler monkey (Alouatta caraya): a study with mycobacterial interspersed repetitive unit–variable number of tandem repeats genetic profiling

In March 2024, faecal samples were collected in the Silesian Zoological Garden in Chorzów from the enclosure inhabited by two black howler monkeys. One was a 15-year-old female from Apenheul Primate Park (in Apeldoorn in the Netherlands), and the second was a 9-year-old male from Wrocław Zoological Garden (in Wrocław in Poland). The animals arrived at the zoo respectively on 21/09/2023 and 16/11/2023. Until 21/01/2024, a third black howler monkey, a 5-year-old female, was in the enclosure. This animal died of intestinal perforation. Above the monkeys’ cage, there is flying space for a female Green araçari (Pteroglossus viridis), which has been kept in the zoo in Chorzów since 27/09/2023 and which came from the Zoological Garden in Łódź in Poland. Some armadillos (Dasypodidae) were also housed in the cage for a very short time. The black howler monkeys had suffered from recurrent diarrhoea since arriving at the zoological garden. The only treatment had been the administration of prebiotics.

To reduce stress for the animals and maintain their well-being, the samples were collected with the zookeeper’s help during the routine cleaning of the enclosure. The faeces were collected from a freshly laid plastic tarpaulin shortly after defecation. The material was placed in a sterile container and then transported to the laboratory in refrigerated conditions (8°C). Part of the material was intended for parasitological tests, which were performed within 24 h of collection, and part was frozen at –20°C for further testing.

The faecal samples were processed by suspension, decontamination and culture, according to the World Organisation for Animal Health (WOAH) Terrestrial Manual 2024, Chapter 3.1 (28). Briefly, 1 g of faeces was shaken in distilled water for 30 min at room temperature. Then, the uppermost 5 mL of the faeces suspension was transferred to a tube containing 20 mL of 0.95% 3-hydroxy-2-phenylcinchoninic acid and incubated for 18 h at room temperature. The undistributed sediment was then inoculated into five types of solid media: Stonebrink, Löwenstein–Jensen, Middlebrook and Herrold’s egg yolk (all from Becton Dickinson, Franklin Lakes, NJ, USA), with and without mycobactin. The media were incubated at 37°C for eight months and checked for colonies weekly.

Antimicrobial susceptibility was tested using broth microdilution. For this purpose, 96-well SLOMYCO Sensititre titration plates (Thermo Fisher Scientific, Waltham, MA, USA) were used, which allow the simultaneous determination of susceptibility to 14 antibiotics. These plates contain freeze-dried antibiotics in a range of concentrations. A test for M. avium was performed as recommended by the Clinical and Laboratory Standards Institute (CLSI) for the bacterium’s susceptibility to clarithromycin, moxifloxacin, amikacin and linezolid (24). At the first stage of the test, an inoculum of a mycobacterial suspension at the optical density of 0.5 McFarland scale was prepared. A 50-μL aliquot of inoculum was transferred to 10 mL of cation-supplemented Mueller–Hinton broth and N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid buffer (Thermo Fisher Scientific) with 5% oleic acid–albumin-dextrose-catalase (OADC). One hundred μL of suspension was pipetted into a 96-well titration plate and incubated at 36°C ± 1°C for 7 d (1, 6). Breakpoint to minimum inhibitory concentrations were interpreted and the strain was classified into one of three groups (susceptible (S), intermediate (I) or resistant (R)) following the CLSI criteria (1, 3).

Isolation of DNA from colonies was undertaken using the Genolyse Isolation Kit (Hain Lifescience, Nehren, Germany). Identifying isolated NTM strains was carried out using the GenoType Mycobacterium CM test (Hain Lifescience). Both kits were used according to the manufacturer’s manuals. The GenoType Mycobacterium CM test is qualitative and is based on DNA-strip technology. The procedure consists of the following steps: mycobacterial DNA isolation from the obtained culture, amplification and reverse hybridisation. The presence of bands at control sites (CC, IC and GC) indicates the correct reaction course and the presence of mycobacteria genetic material.

Isolates of DNA were subjected to IS901, IS900 and IS1245 analysis according to the method previously described by Majchrzak et al. (19) with modifications. This method is used for the rapid identification of M. avium subspecies. A PCR reaction was performed in a total volume of 50 μL containing 20 ng of DNA, 25 μL of Platinum Multiplex PCR Master Mix 2× (Applied Biosystems, Carlsbad, CA, USA) and 50 nM of each primer which was the same as was used by Lalle at al. (18). The following conditions were used: an initial denaturation step at 95°C for 15 min; 28 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 90 s and extension at 72C for 1 min; and a final extension step at 72C for 2 min. The reaction was performed using a T3000 thermal cycler (Analytik Jena, Jena, Germany). Electrophoresis was performed at 70 V (2.4 V/cm) until the bromophenol blue dye reached 6 cm from the wells. The gel was then stained in an ethidium bromide solution (0.5 μg/mL) for 10 min and destained in water for another 10 min. It was visualised under UV light using a FluorChem 8800 system with Alpha EaseFC v. 3.1.2 software (AlphaInnotech, San Leandro, CA, USA). A 100 bp Plus DNA Ladder size marker from MBI Fermentas (Vilnius, Lithuania) was used for all analysed IS types. The predicted sizes of the PCR fragments for IS1245, IS900 and IS901 were 427 bp, 389 bp and 262 bp, respectively.

The typing method used, MIRU-VNTR, is a powerful technique for analysing mycobacteria genotypes and elucidating possible phylogenetic relationships between strains. The isolate underwent multi-locus variable number of tandem repeats analysis (MLVA) employing the eight variable-number tandem-repeat locus scheme proposed by Thibault et al. (26). Primer pairs to amplify the tandem repeat (TR)292, TRX3, TR25, TR47, TR3, TR7, TR10 and TR32 loci were chosen from the work of Cochard et al. (7). The PCR procedure was carefully optimised and performed as described by Majchrzak et al. (19). The size of each amplicon was determined using BioNumerics software v. 4.6 (Applied Maths, Sint-Martens-Latem, Belgium) to calculate motif repeat number. This number code was then matched against those entered in the MAC-INMV-SSR database (7).

The genotypic data of seven loci (MIRU07, 10, 25, 32, 47, 292 and X3) were analysed. As reported by other investigators, many M. avium isolates have not produced PCR products at the TR3 locus. This locus is also known as monomorphic for MAH. Studies involving analysis of seven MIRU-VNTR loci are considered equivalent to the previously reported eight-MIRU-VNTR-locus studies (14). Therefore, we excluded the TR3 locus from further analyses. Clustering analysis of VNTR profiles was performed based on the MST algorithm using the VNTR profiles of the N32 strain and 349 strains isolated from different sources (cattle, pigs, the environment and patients) and different geographical regions (Japan, Finland and France) described in previous reports (14, 31). We used BioNumerics software v. 4.6. A phylogenetic tree was reconstructed by first selecting a categorical coefficient. The priority rule was set so that the type with the highest number of single-locus variants would be linked first, with creation of hypothetical types not permitted (13).

To detect possible concomitant parasitic diseases, a microscopic examination was performed using Faust flotation with zinc sulphate (32). In addition, immunochromatographic tests were carried out to determine the presence of Giardia intestinalis and Cryptosporidium spp. antigens (Stick Crypto-Giardia; OPERON, Zaragoza, Spain). Afterwards, DNA was isolated using a commercial kit (Genomic AX Stool; A&A Biotechnology, Poland, Gdańsk,), and PCR tests were performed for the presence of DNA of intestinal protozoa: G. intestinalis (511 base pair (bp); β-giardin) (18), Cryptosporidium spp. (840 bp; 18S small subunit (SSU) ribosomal RNA (rRNA)) (29), Entamoeba coli (166 bp; SSU rRNA) (23), Pentatrichomonas hominis (339 bp; 18S rRNA) (11). Electrophoresis was performed on a 1.5% agarose gel to reveal the sought genetic material. The positive results were then sent for sequencing to Laboratory of DNA sequencing and synthesis, Institute of Biochemistry and Biophysics, Polish Academy of Sciences in Warsaw.

The material was also tested by using the multiplex PCR method for the presence of Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii DNA (ba, 166 bp; SSU rRNA; 752 bp; SSU rRNA; 580 bp; SSU rRNA), (32). Both, PCR and multiplex PCR tests were performed with commercial mix (StartWarm HS-PCR Mix; A&A Biotechnology, Poland, Gdańsk).

Numerous nonchromogenic, small, round, cream-coloured colonies of fastidious cells developed on all media types in four to six weeks. The isolated strain was classified as a Mycobacterium avium complex subspecies based on the appearance of single colonies on differential media and the GenoType Mycobacterium CM test, and this was confirmed by the IS analysis. The strain showed the presence of IS1245 but not of IS900 or IS901, which allowed its classification as M. avium subsp. hominissuis (Table 1). A new MIRU-VNTR pattern was identified (22221229) and added to the MAC-INMV-SSR database. The tested strain was found to be susceptible to amikacin and clarithromycin, intermediately susceptible to moxifloxacin, and resistant to linezolid (Table 2).

The VNTR eight-locus pattern of strain N32 was determined, and a new pattern was identified. This was the first case of Mycobacterium avium subsp. hominissuis detected in the black howler monkey; therefore, we decided to compare its VNTR profile with other VNTR patterns available in the databases. We compared our data on the seven MIRU-VNTR loci with data obtained from the seven MIRU-VNTR loci from Japanese (human, porcine, bovine and environmental), French (human and porcine) and Finnish (human and porcine) records and created an MST analysis (Fig. 1). The N32 VNTR pattern was a unique orphan type and was located near French and Finnish isolates (human and porcine) and isolates from Japanese pigs.

Fig. 1.

Combined minimum spanning tree analysis based on seven-locus (mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTR) genotyping taking the 07, 10, 25, 32, 47, 292 and X3 loci of Mycobacterium avium subsp. hominissuis (MAH) strains. The N32 MAH strain isolated from two black howler monkeys with recurrent diarrhoea (N32) and previously published strain data were used for the analysis. Circle sizes are proportional to the number of isolates sharing an identical VNTR pattern. MAH 104 is a reference strain. Lines connecting two types denote single- or double-locus variations with each length

In microscopic examination, the only parasites observed were cysts resembling Giardia intestinalis or trichomonas pseudocysts. Because their morphological forms were similar and it was possible for both protozoa to occur in black howler monkeys, it was decided to perform more advanced tests. The immunochromatographic stick test was negative for G. intestinalis and Cryptosporidium spp. antigens. After performing PCRs and electrophoresis, a positive result was obtained only for the presence of the DNA of G. intestinalis. Sequencing resulted in assemblage B (data not shown).