Isolation and genetic analysis of mycobacteria from suspect tuberculous lesions in slaughtered cattle from Wolaita, Ethiopia
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Bovine tuberculosis (bTB), caused by Mycobacterium bovis and other members of the Mycobacterium tuberculosis complex (MTBC), is a significant concern for livestock and public health in Ethiopia. This study aimed to isolate and genetically characterize the causative agents of bTB in cattle from four abattoirs in the Wolaita zone of Ethiopia. A total of 2,251 cattle were examined post-mortem, and suspect tuberculous lesions were identified in 122 animals. From these animals, 180 tissue samples were collected and processed for bacteriological culture and genetic analysis, including the Loopamp ™ commercial loop-mediated isothermal amplification kit, PCR targeting RD4 and RD9 loci and whole-genome sequencing (WGS). Bacteriological culture using mycobacteria growth indicator tube and Lowenstein–Jensen media ultimately identified 18 culture-positive samples, with WGS confirming M. bovis in lesions from four animals and M. tuberculosis in lesions from one animal. The M. bovis and M. tuberculosis isolates showed genetic similarity to previously identified MTBC lineages in Ethiopia. The presence of M. tuberculosis in cattle raises concerns about human-to-animal transmission. Additionally, non-tuberculous mycobacteria were isolated from lesions from multiple animals. Our study genetically characterized bacteria from suspect tuberculous lesions and provides the research community with new genome data for Ethiopian isolates of M. bovis and M. tuberculosis .
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Dear Dr Zaba, Thank you for your revised submission. I'm pleased to inform you that the manuscript has now been accepted. Congratulations and please consider ACMI again for your future submissions. Best wishes, John.
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Thank you for your submission. The manuscript has been reviewed by two independent reviewers and both agree the manuscript is a valuable contribution and only requires minor amendments before it can be accepted for publication. Please address the reviewers comments below by the return date below. Best wishes, John.
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Comments to Author
In this manuscript, "Isolation and genetic analysis of mycobacteria from suspect tuberculous lesions in slaughtered cattle from Wolaita, Ethiopia", the authors collected tissue samples from cattle slaughtered in abattoirs in the Wolaita zone of Ethiopia. The 18 isolates that tested positive for members of the Mycobacterium tuberculosis complex were sequenced and the resulting genomes were compared to other genomes previously collected in Ethiopia and elsewhere. The manuscript is well written, the analysis appropriate and the results will be of interested to those researchers investigating TB in Ethiopia. I have no major concerns with the paper. My minor comments are as follows: Methods - It's unclear how the subset of contextual Ethiopian genomes analyses alongside the genomes generated as part of …
Comments to Author
In this manuscript, "Isolation and genetic analysis of mycobacteria from suspect tuberculous lesions in slaughtered cattle from Wolaita, Ethiopia", the authors collected tissue samples from cattle slaughtered in abattoirs in the Wolaita zone of Ethiopia. The 18 isolates that tested positive for members of the Mycobacterium tuberculosis complex were sequenced and the resulting genomes were compared to other genomes previously collected in Ethiopia and elsewhere. The manuscript is well written, the analysis appropriate and the results will be of interested to those researchers investigating TB in Ethiopia. I have no major concerns with the paper. My minor comments are as follows: Methods - It's unclear how the subset of contextual Ethiopian genomes analyses alongside the genomes generated as part of this study were chosen. Please could you clarify why these genomes were included? - Representative genomes for some (not all) of the MTBC lineages were included alongside the two MTB genomes identified in this study. Why were these genomes included? There is no information on these in the methods. Additionally, why were some of the other lineages (L3, L8-L10) not included in the analysis? I would repeat the analysis with these additional genomes and root the tree in Figure 4 with L8 to give a better representation of the population structure of the MTBC (rooting with the L4 H37Rv reference is not ideal for rooting the MTBC). I'm not sure why five BCG genomes were included. One should be sufficient. Figures - Figure 3. It would be helpful to add the bovis lineage as a metadata strip. TB-profiler can assign the Zwyer lineages to these genomes which is what I would recommend. Could the sample names generated as part of this study be highlighted in bold to make them easier to see in the tree. The headers in the legends are very small, please make them bigger. I presume the scale at the bottom is in SNPs? Please state this in the legend and make the numbers bigger. - Figure 4. See comment above regarding the inclusion of the missing lineages and rooting the phylogenetic tree. Also the comments about Figure 3 regarding text size and the SNP scale apply here.
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Very good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
The authors of this manuscript have studied more than 2000 cattle carcasses from 4 abattoirs in Ethiopia and have processed 180 tissue samples from lymph nodes, lungs and livers for bacteriological culture and genetic analysis, using different mycobacterial identification techniques including a commercial LAMP (loop-mediated isothermal amplification) kit, PCR tests focused on deleted regions RD9 and RD4, and whole genome sequencing. The authors show that 5 cattle harbored mycobacteria from the M. tuberculosis complex (MTBC) , whereby multiple samples per animal tested positive. There were 4 animals that had carried M. bovis and interestingly also one where M. tuberculosis was identified in two independent samples. The authors also report that many other non-tuberculous mycobacteria (NTM) of …
Comments to Author
The authors of this manuscript have studied more than 2000 cattle carcasses from 4 abattoirs in Ethiopia and have processed 180 tissue samples from lymph nodes, lungs and livers for bacteriological culture and genetic analysis, using different mycobacterial identification techniques including a commercial LAMP (loop-mediated isothermal amplification) kit, PCR tests focused on deleted regions RD9 and RD4, and whole genome sequencing. The authors show that 5 cattle harbored mycobacteria from the M. tuberculosis complex (MTBC) , whereby multiple samples per animal tested positive. There were 4 animals that had carried M. bovis and interestingly also one where M. tuberculosis was identified in two independent samples. The authors also report that many other non-tuberculous mycobacteria (NTM) of different species were found in the lesions and that in some samples both MTBC and NTM were found. These data are all interesting to be reported. While the identification of M. bovis in 4bcattle carcasses indicates that there is still contamination of cattle ongoing in the region, the identification of M. tuberculosis in two samples from one animal is intriguing, especially because M. tuberculosis was reported to be attenuated in cattle in at least two previous papers. I would suggest to the authors to discuss this fact in more detail and provide a possible explanation (speculation) for this finding, and discuss it in the light of the literature existing on this subject. Overall the work appears to be carefully carried out and the results are well presented and clearly discussed. Apart from the request above I do not have any other points that might improve the manuscript.
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Very good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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