Molecular screening finds no trace of dangerous mycobacteria in Algerian milk

Milk is one of the most widely consumed animal products in the world, and in many regions it still reaches consumers raw or only lightly processed. That tradition carries a hidden risk, because several members of the bacterial genus Mycobacterium can be shed into milk by infected animals and are capable of causing serious disease in people. In Algeria, where bovine tuberculosis is considered enzootic and serological evidence of infection has already been reported in eastern cattle herds, the question of what actually circulates in the dairy supply is far from academic. A new study has now addressed that question directly, using highly sensitive molecular methods to screen hundreds of milk samples from farms in northern Algeria for five pathogenic mycobacterial species, and the results offer a measure of reassurance alongside important caveats about what a negative result can and cannot mean.

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