The research, conducted by a team spanning Algeria and the IHU Méditerranée Infection in Marseille, France, focused on the Béjaïa region of northern Algeria. Between December 2023 and January 2024, the investigators collected 160 bovine and 231 caprine milk samples, a total of 391 specimens, drawn during routine milking from seven farms. The samples were transported at 4 degrees Celsius and shipped to Marseille under import permit ER-02-2024 for analysis. The choice of targets reflected both local and global concerns: the Mycobacterium tuberculosis complex, which includes Mycobacterium bovis, the agent of bovine tuberculosis; Mycobacterium uberis, a recently described species that causes nodular thelitis and tuberculoid scrotitis in livestock; Mycobacterium leprae and Mycobacterium lepromatosis, the two leprosy bacilli, whose animal reservoirs have been documented outside North Africa; and Mycobacterium ulcerans, the cause of Buruli ulcer, which has been reported in domestic animals elsewhere.

The laboratory workflow was designed to maximise sensitivity while guarding against the two classic pitfalls of molecular diagnostics: failed extraction and contamination. Each milk sample was first centrifuged at 3,000 times gravity for 15 minutes to concentrate bacteria and cellular debris into a pellet. A synthetic internal amplification control, designated TIIS, was then added to every pellet before automated DNA extraction on a QIAGEN EZ1 Advanced XL platform. This internal control serves a critical function: if it amplifies in the downstream polymerase chain reaction, it demonstrates that DNA was successfully extracted and that no inhibitors in the milk matrix suppressed the enzymatic reaction. Without such a control, a negative result would be ambiguous, because it could reflect either a true absence of the pathogen or simply a technical failure.

Detection relied on five independent hydrolysis-probe quantitative PCR assays, each targeting a species-specific genetic signature. The RLEP assay targeted M. leprae, the RLPM assay targeted M. lepromatosis, an assay directed at the ESX-1 locus detected M. uberis, a CRISPR-Csm4-based assay identified the M. tuberculosis complex, and a PPE-targeted assay screened for M. ulcerans. Thermocycling consisted of an initial denaturation step at 95 degrees Celsius for two minutes, followed by 45 cycles of 95 degrees Celsius for 15 seconds and 58 degrees Celsius for 60 seconds. A specimen was scored as positive only when a target-specific amplification curve crossed the fluorescence threshold within the 45-cycle run, and only if the corresponding internal control amplified and the no-template water control remained negative. Samples without target-specific amplification were recorded as having undetermined cycle threshold values.

Contamination control received particular attention. Positive control materials were deliberately excluded from routine sample-testing runs to minimise the risk of cross-contamination, a precaution that is especially important when screening for organisms such as M. leprae, where even trace carryover of amplifiable DNA could produce a false positive. The external M. leprae positive control was tested separately, and additional validation assays were performed to confirm the specificity of each primer-probe set and the overall performance of the qPCR platform. The published analytical sensitivities of the RLEP and RLPM assays, along with the oligonucleotide sequences and cycle threshold interpretation criteria, were documented in the supplementary materials accompanying the paper, allowing other laboratories to reproduce or compare the methodology.

The results were unambiguous. All TIIS internal amplification controls amplified successfully, confirming that DNA extraction worked in every one of the 391 samples and that no qPCR inhibition was detectable. All no-template water controls remained negative, providing no evidence of reagent contamination. Yet no target-specific amplification was observed in any sample for any of the five mycobacterial targets. In other words, across every bovine and caprine milk specimen tested, the investigators could not detect DNA from the tuberculosis complex, from either leprosy bacillus, from M. uberis, or from M. ulcerans.

With zero detections, the study turned to statistics to quantify the strength of that negative finding. Using exact one-sided 95 percent confidence limits, the researchers calculated that the upper bound for the proportion of qPCR-positive samples was 1.85 percent in bovine milk, 1.29 percent in caprine milk, and 0.76 percent overall. These figures are worth understanding correctly: they do not mean that 1.85 percent of samples were contaminated, but rather that, given 391 samples with no detections, the true proportion of positive samples is statistically very unlikely to exceed those thresholds. The limits describe detectable DNA in the milk itself, not the prevalence of infection in the source animals, a distinction that matters greatly for interpreting the public-health significance of the work.

The authors are careful to place these findings in context. The absence of M. tuberculosis complex DNA is reassuring, but it should not be read as proof that bovine tuberculosis is absent from the region. Mycobacterial shedding in milk can be intermittent, meaning that a single point-in-time sample may miss an animal that sheds bacteria at other moments. The survey did not specifically select animals with mastitis, tuberculosis-compatible lesions, or known positive herd status, which are precisely the animals most likely to shed pathogens into milk. Sampling was also restricted to seven farms in a single region, so the results cannot be extrapolated to all of Algeria. Bacterial loads below the analytical sensitivity of the assays remain possible, and qPCR alone cannot establish whether any detected organism would be viable; culture and herd-level testing remain essential complements. Published milk studies show marked variation in M. bovis detection depending on herd infection status, specimen type, and analytical method, with an international meta-analysis reporting a pooled prevalence of around 5 percent in individual samples, a figure that underscores how heterogeneous the global picture is.

The same caution applies to the other four targets. Non-detection of M. leprae, M. lepromatosis, M. uberis, and M. ulcerans in milk does not exclude their presence in other tissues, other hosts, or environmental compartments. Red squirrels in the British Isles, for example, have been found to harbour leprosy bacilli, and M. ulcerans infection has been documented in domestic animals in West Africa, demonstrating that these organisms can occupy unexpected ecological niches. Nevertheless, the absence of target-specific amplification across 391 internally controlled extracts provides a valuable regional baseline for northern Algeria, a baseline that simply did not exist before.

The study’s broader significance lies in its methodology as much as its results. By combining rigorous internal amplification controls, strict contamination management, and statistically framed interpretation of zero detections, the work offers a template for how molecular surveillance of the dairy supply should be conducted in regions where raw milk consumption is common and veterinary diagnostic infrastructure may be limited. The authors conclude that broader longitudinal studies, combining repeated milk sampling with culture, tissue analysis, and herd-level diagnostics, are required to define the occurrence and public-health relevance of pathogenic mycobacteria in Algerian dairy systems. Until such studies are completed, the new findings stand as a carefully qualified but genuinely encouraging data point: at the time of sampling, no molecular evidence indicated that milk from the surveyed herds carried detectable DNA from any of five dangerous mycobacterial species.

Subject of Research: Molecular detection of zoonotic pathogenic mycobacteria in bovine and caprine milk in northern Algeria

Article Title: Absence of molecular detection of zoonotic mycobacteria in bovine and caprine milk in northern Algeria.

Article References: Souguel, S., Ndiaye, A., Boualam, M., Grine, G., Agag, S., Drancourt, M., & Tazerart, F. (2026). Absence of molecular detection of zoonotic mycobacteria in bovine and caprine milk in northern Algeria.. New Microbes and New Infections, Article 101866. https://doi.org/10.1016/j.nmni.2026.101866

Image Credits: AI Generated

DOI: 10.1016/j.nmni.2026.101866

Keywords: Mycobacterium bovis, Mycobacterium leprae, Mycobacterium ulcerans, Mycobacterium uberis, bovine tuberculosis, raw milk, qPCR, Algeria, food safety, zoonoses, dairy surveillance, molecular diagnostics

The scale of the investigation reflects the difficulty of working with organisms that are ubiquitous in soil and water. The researchers analyzed 193 clinical samples drawn from five distinct sources: 101 fecal samples, 21 nasal swabs, 13 trans-tracheal washes, 30 tissue specimens, and 28 blood samples. Each sample was processed through two parallel routes, direct polymerase chain reaction and culture-based isolation, a dual strategy designed to capture both organisms present in the sample at the time of collection and those capable of growing under laboratory conditions. Decontamination steps, essential when dealing with samples teeming with faster-growing environmental flora, were carefully applied to give the slow-growing mycobacteria a chance to emerge on culture media. The approach mirrors established diagnostic protocols for mycobacterial recovery from clinical material, where the balance between eliminating contaminants and preserving viable target organisms is notoriously delicate.

Identification proceeded in a tiered molecular fashion. Genus-level confirmation relied on PCR targeting the hsp65 gene, a workhorse marker in mycobacterial taxonomy that encodes a heat shock protein and allows discrimination of mycobacteria from other acid-fast organisms. Once the isolates were confirmed as members of the genus Mycobacterium and distinguished from the Mycobacterium tuberculosis complex, species-specific PCR assays were deployed to pin down exactly which non-tuberculous species had been recovered. This hierarchical approach, from broad genus screening to species-level resolution, is critical because the clinical and epidemiological significance of non-tuberculous mycobacteria varies enormously from one species to the next. In total, the workflow yielded eighteen confirmed non-tuberculous mycobacterial isolates, a modest but workable collection for downstream genetic analysis.

The species picture that emerged was strikingly lopsided. Mycobacterium vaccae accounted for two-thirds of the isolates, representing 66.7 percent of the confirmed collection. This organism, first described decades ago and long studied for its immunomodulatory properties, is a common inhabitant of soil, water, and dust, and its dominance in bovine samples underscores how readily environmental mycobacteria colonize or transit through livestock. The remaining isolates comprised other non-tuberculous species, though M. vaccae clearly set the tone of the collection. For veterinary diagnosticians, this predominance matters: when a bovine sample yields acid-fast organisms or tuberculosis-like lesions, the probability that the culprit is an environmental species rather than Mycobacterium bovis has direct consequences for how the finding is interpreted within national tuberculosis control frameworks.

With the isolates in hand, the team turned to the central question of the study: what resistance-associated genes do these bovine non-tuberculous mycobacteria carry? Four genes were selected for screening, each with a well-characterized role in drug resistance in Mycobacterium tuberculosis. The rpoB gene, encoding the beta subunit of RNA polymerase, is the principal target of rifampicin, and mutations within its resistance-determining region are the classic molecular signature of rifampicin resistance. The katG gene encodes catalase-peroxidase, the enzyme that activates the prodrug isoniazid, and disruptions in katG are strongly associated with high-level isoniazid resistance. The rpsL gene, encoding ribosomal protein S12, is implicated in streptomycin resistance, while gyrA, encoding the A subunit of DNA gyrase, harbors mutations in its quinolone resistance-determining region that confer fluoroquinolone resistance. Screening these four targets across the eighteen isolates provided a snapshot of the genetic resistance landscape in bovine non-tuberculous mycobacteria from the region.

The screening results revealed a clear hierarchy. The rpoB gene was the most frequently detected, present in 88.9 percent of the isolates, and its detection was mainly associated with Mycobacterium vaccae. The katG gene turned up in 16.7 percent of isolates, and the gyrA gene in just 5.6 percent, and notably both of these were found exclusively in M. vaccae. The rpsL gene, by contrast, was not amplified in any isolate, suggesting that streptomycin-associated genetic determinants, at least as targeted by the primers used, are absent from this collection. The pattern indicates that within this bovine sample set, genetic elements associated with resistance cluster heavily in one environmental species, raising questions about whether M. vaccae’s ecological lifestyle, including prolonged exposure to antimicrobial residues in agricultural environments, might shape its genetic repertoire.

Sequencing of selected amplicons added an important layer of nuance. When the researchers examined the hotspots of rpoB and katG, the regions where resistance-conferring mutations most commonly occur in Mycobacterium tuberculosis, they found no mutations at all. This is a meaningful negative result: the mere presence of a gene does not imply the presence of resistance-conferring variants within it. However, the story differed for gyrA. Sequence analysis of the quinolone resistance-determining region of gyrA revealed non-synonymous substitutions, meaning nucleotide changes that alter the encoded amino acid sequence of DNA gyrase. Whether these substitutions affect fluoroquinolone susceptibility in these isolates remains an open question, but their presence in the canonical resistance region of the enzyme is exactly the kind of finding that warrants closer phenotypic scrutiny.

The authors are careful, and rightly so, about what these results do and do not demonstrate. The detection of resistance-associated genes and even of amino acid substitutions reflects genotypic variation only. Because drug susceptibility testing was not performed on the isolates, the study cannot confirm phenotypic resistance to rifampicin, isoniazid, streptomycin, or fluoroquinolones. This distinction is fundamental in mycobacteriology. Genes such as rpoB and katG are essential housekeeping and metabolic genes present in virtually all mycobacteria; their detection by PCR says nothing about function unless the specific mutational hotspots are altered. Moreover, resistance mechanisms in non-tuberculous mycobacteria differ in important ways from those in the tuberculosis complex, involving intrinsic factors such as impermeable cell walls, efflux pumps, and species-specific enzymatic machinery, which means that extrapolating interpretive criteria from M. tuberculosis to environmental species is fraught with uncertainty.

Nevertheless, the study carries practical implications for animal health and public health alike. Non-tuberculous mycobacteria can produce tuberculosis-like lesions in bovines, and such lesions can trigger false-positive reactions in tuberculin skin testing, the backbone of bovine tuberculosis surveillance programs. Every misattributed lesion represents a potential misdiagnosis, with consequences ranging from unnecessary slaughter to missed transmission of true Mycobacterium bovis infection. By documenting which non-tuberculous species circulate in Indian cattle and buffaloes, and by beginning to map their resistance gene content, the researchers are building the species-level baseline that surveillance programs need to distinguish environmental noise from genuine tuberculosis signals. The finding that M. vaccae dominates the isolates gives diagnosticians a concrete expectation about what they are most likely to encounter when non-tuberculous organisms appear in bovine material.

The road forward, as the authors indicate, runs through phenotypic and species-specific analyses. Establishing the functional significance of the gyrA substitutions observed in M. vaccae will require correlating genotypes with measured minimum inhibitory concentrations of fluoroquinolones, and ideally with functional studies of the mutant gyrase enzymes. Broader panels of resistance genes, and ultimately whole-genome sequencing, could reveal the full complement of intrinsic and acquired resistance determinants in these organisms, an approach increasingly advocated in the literature on non-tuberculous mycobacterial susceptibility prediction. For now, the study stands as a careful first census: eighteen isolates, one dominant species, one frequently detected resistance-associated gene without hotspot mutations, and a handful of amino acid changes in a quinolone target that demand follow-up. In a world where antimicrobial resistance knows no boundaries between human clinics, farms, and soil, even the humblest environmental mycobacterium from a cow’s gut deserves that kind of attention.

Subject of Research: Molecular identification of non-tuberculous mycobacteria from bovine clinical samples and profiling of resistance-associated genes

Article Title: Molecular identification and resistance-associated gene profiling of non-tuberculous mycobacteria isolated from bovine clinical samples

Article References: Panigrahi, B., Narang, D., Chandra, M., Narang, A., Gupta, K., & Singh, S. T. (2026). Molecular identification and resistance-associated gene profiling of non-tuberculous mycobacteria isolated from bovine clinical samples. Molecular Biology Reports, 53(1), Article 1640. https://doi.org/10.1007/s11033-026-12814-7

Image Credits: AI Generated

DOI: 10.1007/s11033-026-12814-7

Keywords: non-tuberculous mycobacteria, Mycobacterium vaccae, bovine tuberculosis, rpoB, katG, gyrA, antimicrobial resistance, PCR, cattle, diagnostics, genotypic variation, India