The aim of this pilot study was to determine whether M. bovis shedding in buffalo oronasal secretions could be detected by using oronasal swabs, stored in a commercially available nucleic acid stabilising, pathogen inactivating transport media (PrimeStore MTM) with the rapid Xpert MTB/RIF Ultra assay. In a cohort of 12 culture-confirmed M. bovis infected buffaloes, each with at least one positive antigen-specific immunological test result (SCITT, IPRA, IGRA), 5 individuals had an Ultra positive oronasal swab result, which supports the hypothesis that these were infected animals shedding mycobacteria. Although environmental contamination of oronasal secretions with MTBC could not definitively be ruled out, the isolation of M. bovis from head and thoracic lymph nodes from four of five Ultra positive buffaloes, along with a documented immunological response, suggests that the animals were truly infected and the source of the MTBC DNA. Moreover, the three M. bovis exposed (although culture and immunological test negative) buffaloes shared the same environment as the rest of the HiP cohort, and were culled and sampled under the same circumstances, but had negative Ultra results, indicating that the detection of MTBC DNA in the five Ultra positive buffaloes were not due to environmental contamination.
The results in this study further demonstrated that the method of collection and inactivation using PrimeStore MTM to store oronasal swab samples did not interfere with detection of MTBC DNA using the Ultra assay. A previous study assessed the use of PrimeStore MTM with swabs collected from buffalo tissue samples to detect MTBC DNA by Ultra; this was found to be a safe, easy, and effective method to transport samples at room temperature to screen animals for the presence of M. bovis 13. The current study has shown that PrimeStore MTM can also be used to preserve MTBC DNA in oronasal swabs that, when used with the Ultra assay, provides a simple method for detecting MTBC DNA in oronasal secretions, making it a promising assay for evaluating shedding.
All buffaloes (excluding the three buffaloes that were culture and immunological test negative) were culled, based on their positive antigen-specific immunological test results (SCITT, IGRA and/or IPRA), which provided an indirect indication of infection. Twelve buffaloes were confirmed to be M. bovis infected by mycobacterial culture from tissues. However, other studies suggest that culture is an imperfect method for detection of M. bovis infection 2,10. Conventional mycobacterial culture uses a relatively harsh decontamination treatment and samples may be subjected to freeze-thaw cycles prior to culture, both of which may reduce the viability of mycobacteria that may be present in small numbers in oropharyngeal swabs 15. Mycobacterial culture also has poor sensitivity and takes up to 56 days for adequate growth; therefore, culture of swabs may be a poor tool to assess shedding in buffalo herds. However, use of a highly sensitive qPCR assay (Ultra), in combination with samples stored in PrimeStore MTM, which stabilises the nucleic acids in the swab samples, may facilitate positive identification of paucibacillary MTBC DNA in oronasal secretions, which would increase the proportion of test-positive infected buffaloes.
Four of five buffaloes, in which M. bovis shedding in oropharyngeal secretions was detected by Ultra, had M. bovis culture-positive results from head and thoracic lymph nodes. One animal (B64), in which MTBC DNA shedding was also detected, had generalised M. bovis culture-positive lesions in the abdominal serosa, indicative of extrapulmonary bTB. Studies have found that the distribution and progression of lesion development are correlated to the route of mycobacterial infection 16. For example, cattle, infected endotracheally with M. bovis, had culture-positive gross pathological lesions in the lungs and thoracic cavity, whereas those that were orally infected had lesions in abdominal tissues 16. Gastrointestinal lesions may also be caused by swallowing infectious respiratory secretions or through lymphatic spread 17,18. Although only a single case, the Ultra result from buffalo B64 suggests that buffaloes with extrapulmonary bTB may also be capable of shedding. Moreover, even though the anatomical location of lesions provides some information on possible routes and sites of infection, it cannot be used to definitively determine whether the individual is infectious and shedding. For example, M. bovis was cultured from faeces in cattle that only had bTB lesions in thoracic tissues 17. Therefore, further studies on the pathways of M. bovis excretion in buffaloes are required.
One of the limitations of the study is that it could not be definitively determined if detection of MTBC DNA in oronasal swabs was a result of shedding or environmental contamination. Positive antigen-specific immunological test results in Ultra assay positive buffaloes provide further evidence that positive Ultra results were found in truly infected buffaloes. In addition, the high specificity of the Ultra assay was supported by the negative results from historically bTB negative buffalo herds, which were in environments that may contain environmental mycobacteria. Therefore, these results indicate that oronasal swabs with a positive Ultra result were detecting MTBC DNA, rather than nontuberculous mycobacteria. However, future studies should include oronasal swabs from larger cohorts of M. bovis uninfected buffaloes in bTB endemic environments to determine if the Ultra assay can detect oronasal contamination with MTBC.
An additional limitation was that since the swabs were stored in PrimeStore MTM, which inactivates microorganisms, the swabs could not be cultured and therefore direct verification of the Ultra results by determining the presence of viable bacilli could not be performed. Finally, only a single swab was collected from each buffalo, which represents a single time point. If the buffaloes were serially sampled over time, more animals may have been detected as shedding in this cohort of infected buffaloes.
In summary, this pilot study supports the use of the Ultra assay with oronasal swabs to screen for shedding in M. bovis infected buffaloes, which may advance our knowledge of the epidemiology of bTB. Since oronasal swabs were stored in PrimeStore MTM, all microorganisms, including viral and bacterial pathogens, were inactivated, which would potentially permit movement of samples without risk of accidental pathogen spread. PrimeStore MTM stabilises DNA at room temperature, which also facilitates field collection and processing prior to performing the Ultra assay. Since the Ultra equipment is portable and can be easily operated without extensive training, the next step would be to determine if this is a field-friendly screening tool. The oronasal swab Ultra assay provides a more rapid ante-mortem test result than culture (which typically takes 56 days) to confirm the presence of MTBC. Based on the relatively large proportion of shedders that were detected in our cohort, future work should include serial monitoring of shedding in a group of M. bovis infected buffaloes to evaluate disease transmission risk under different conditions to improve management and understanding of the epidemiology of bTB in wildlife.