East Mediterranean Lineage of Brucella melitensis in Human Isolates and Milk Samples in Oman Using MLVA-14

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Studies about genetic diversity of Brucella are limited in the country. This study aimed to genotype Brucella melitensis in human isolates and milk samples using multi-locus variable number tandem repeats analysis (MLVA-14) in Oman. Methods MLVA-14 was employed for forty-nine B. melitensis recovered from human isolates (n = 21), one goat isolate, and milk samples (n = 27). Results Clustering analysis separated the 49 B. melitensis strains into two main clusters including 31 genotypes. In Dhofar Governorate, shared genotypes among different animal species were identified; the same genotypes were found also in human isolates. Moreover, there was a close genetic relationship between human and milk sample strains from Dhofar and AD Dakhiliya Governorates. Phylogeography investigated by Minimum Spanning Tree analysis showed that Omani strains belonged to the East Mediterranean lineage and formed a distinct branch with a close relationship with two strains from the United Arab Emirates. Moreover, eight Omani strains genotyped from milk shared the same MLVA profile as strains from Spain, Portugal, China, India, and Turkey. The caprine isolate was an outlier correlated with a big cluster mostly formed by isolates from China with other strains from Portugal, Kazakhstan, Turkey, Mongolia, Marocco, France and Spain. Conclusions This study highlights the zoonotic nature of B. melitensis transmission from infected livestock to humans and also its circulation among different animal species. The One Health approach is the way to develop policies and programs for disease surveillance and control. 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F1000Research 2025, 14 :191 ( https://doi.org/10.12688/f1000research.161111.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details Export Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente EXPORT Select a format first Track Share ▬ ✚ Research Article East Mediterranean Lineage of Brucella melitensis in Human Isolates and Milk Samples in Oman Using MLVA-14 [version 1; peer review: 1 approved with reservations] Khalsa Altoubi https://orcid.org/0000-0001-7018-0244 1,2 , Zakariya Al Muharrmi 3 , Waleed AlMarzooqi https://orcid.org/0000-0002-6840-7224 2 , [...] Salma Al Adwani https://orcid.org/0000-0001-6069-7768 2 , Kaadhia Al Kharousi 2 , Shytyrbayeva Zamzagul Abdildaevna 4 , Simone Peletto https://orcid.org/0000-0002-8684-898X 5 , Yasmin ElTahir https://orcid.org/0000-0002-0246-826X 2 Khalsa Altoubi https://orcid.org/0000-0001-7018-0244 1,2 , Zakariya Al Muharrmi 3 , [...] Waleed AlMarzooqi https://orcid.org/0000-0002-6840-7224 2 , Salma Al Adwani https://orcid.org/0000-0001-6069-7768 2 , Kaadhia Al Kharousi 2 , Shytyrbayeva Zamzagul Abdildaevna 4 , Simone Peletto https://orcid.org/0000-0002-8684-898X 5 , Yasmin ElTahir https://orcid.org/0000-0002-0246-826X 2 PUBLISHED 12 Feb 2025 Author details Author details 1 Central Laboratory for Animal Health, Department of Animal Diseases Diagnosis, Oman, Muscat, Oman 2 Animal & Veterinary Sciences, Sultan Qaboos University, Muscat, Muscat Governorate, Oman 3 Microbiology & Immunology, Sultan Qaboos University, Muscat, Muscat Governorate, Oman 4 Biological Safety, Kazakh National Agrarian Research University, Almaty, Kazakhstan 5 Istituto Zooprofilattico Sperimentale del Piemonte, Turin, Italy Khalsa Altoubi Roles: Investigation, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing Zakariya Al Muharrmi Roles: Investigation, Methodology, Supervision Waleed AlMarzooqi Roles: Investigation, Methodology, Supervision Salma Al Adwani Roles: Supervision, Writing – Review & Editing Kaadhia Al Kharousi Roles: Investigation, Methodology Shytyrbayeva Zamzagul Abdildaevna Roles: Software, Writing – Review & Editing Simone Peletto Roles: Investigation, Methodology, Writing – Review & Editing Yasmin ElTahir Roles: Investigation, Methodology, Project Administration, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing OPEN PEER REVIEW DETAILS REVIEWER STATUS This article is included in the Pathogens gateway. Abstract Background Brucellosis is the most common zoonotic disease in Oman. Studies about genetic diversity of Brucella are limited in the country. This study aimed to genotype Brucella melitensis in human isolates and milk samples using multi-locus variable number tandem repeats analysis (MLVA-14) in Oman. Methods MLVA-14 was employed for forty-nine B. melitensis recovered from human isolates (n = 21), one goat isolate, and milk samples (n = 27). Results Clustering analysis separated the 49 B. melitensis strains into two main clusters including 31 genotypes. In Dhofar Governorate, shared genotypes among different animal species were identified; the same genotypes were found also in human isolates. Moreover, there was a close genetic relationship between human and milk sample strains from Dhofar and AD Dakhiliya Governorates. Phylogeography investigated by Minimum Spanning Tree analysis showed that Omani strains belonged to the East Mediterranean lineage and formed a distinct branch with a close relationship with two strains from the United Arab Emirates. Moreover, eight Omani strains genotyped from milk shared the same MLVA profile as strains from Spain, Portugal, China, India, and Turkey. The caprine isolate was an outlier correlated with a big cluster mostly formed by isolates from China with other strains from Portugal, Kazakhstan, Turkey, Mongolia, Marocco, France and Spain. Conclusions This study highlights the zoonotic nature of B. melitensis transmission from infected livestock to humans and also its circulation among different animal species. The One Health approach is the way to develop policies and programs for disease surveillance and control. READ ALL READ LESS Keywords Brucella melitensis, MLVA-14, genetic diversity, Oman. Corresponding Author(s) Yasmin ElTahir ( [email protected] ) Close Corresponding author: Yasmin ElTahir Competing interests: No competing interests were disclosed. Grant information: This study was supported by the research council (RC/RG-AGR/ANVS/21/01). The funders had no role in the design of the study, data collection, analyses, decision to publish or preparation of the manuscript. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2025 Altoubi K et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite: Altoubi K, Al Muharrmi Z, AlMarzooqi W et al. East Mediterranean Lineage of Brucella melitensis in Human Isolates and Milk Samples in Oman Using MLVA-14 [version 1; peer review: 1 approved with reservations] . F1000Research 2025, 14 :191 ( https://doi.org/10.12688/f1000research.161111.1 ) First published: 12 Feb 2025, 14 :191 ( https://doi.org/10.12688/f1000research.161111.1 ) Latest published: 13 Mar 2026, 14 :191 ( https://doi.org/10.12688/f1000research.161111.2 )  There is a newer version of this article available. Suppress this message for one day. Introduction Brucellosis is a zoonotic disease affecting human and animal species, including livestock, wild animals, and marine mammals. In animals, it causes abortion, mastitis, reproductive disorders, and reduced milk production. In human, brucellosis can cause various symptoms, from a mild flu-like illness to severe complications such as arthritis and endocarditis, making early detection and control critical. The disease is considered as a main public health concern due to its impact on livestock productivity and human health. 1 – 3 In Oman, brucellosis remains the most common zoonosis. The disease is endemic in Dhofar Governorate due to its humid climate, especially during the monsoon season. A common practice of keeping animals, such as cattle, sheep, and goats, in proximity significantly increases the chances of Brucella spillover. Moreover, human-to-animal contact increases the risk of transmission, necessitating robust surveillance and control measures. 4 – 6 Brucella is an intracellular Gram-negative coccobacillus bacterium. There are various Brucella species. The classic known six species are B. abortus , B. melitensis , B. ovis , B. canis , B. suis , and B. neotomae. 7 More species were recognized later like, B. ceti , B. pinnipedialis , B. microti , B. inopinata , B. papionis , and B. vulpis. 8 – 10 Brucella melitensis has been widely reported in humans and livestock in Oman. 4 , 6 Brucella can be isolated by culture and identified by serological tests and molecular assays. 11 It can also be identified according to CO 2 requirements and biochemical tests such as H 2 S production, urease activity, and agglutination with monospecific sera (A and M). Further identification can be achieved using selective media containing dyes such as thionin or basic fuchsin and phage typing. Brucella isolation is the most reliable method for diagnosis but it is time-consuming and needs special facilities. 12 Rose Bengal plate test is a rapid test for screening Brucella antibodies in both livestock and humans. 13 A milk ring test is utilized to identify Brucella antibodies in cow’s milk. 14 Moreover, another screening test is the Brucella rapid test which can be used for various field samples including blood, plasma, serum, and milk. 15 Enzyme-linked immunosorbent Assays (ELISA) have been widely used as a confirmatory test. 16 The Brucella genome encodes virulence factors and metabolic pathways crucial for intracellular survival. Brucella species show similarity higher than 90% at the genome level, making it difficult to differentiate between Brucella strains through conventional genetic analysis techniques. 17 – 19 Molecular methods are widely used for Brucella diagnosis such as conventional PCR, real-time PCR, multiplex PCR, multiple loci variable number tandem repeat analysis (MLVA), and single nucleotide polymorphism (SNP) analysis. Conventional PCR involves DNA amplification of a single target (singleplex PCR) or multiple targets (multiplex PCR). AMOS and Bruce-ladder are multiplex PCR methods that amplify multiple Brucella target genes in a single reaction. These assays use multiple sets of primers, each specific to a different region in the genome, to differentiate between Brucella species. 20 – 23 MLVA is widely used to identify Brucella genetic diversity. A tandem repeat is a sequence of two or more DNA base pairs repeated and directly adjacent to each other in the genome. These repeats can vary in length and number. MLVA can be used in various genetic studies, including genetic fingerprinting, studying genetic diversity, and identifying hereditary diseases. By analyzing tandem repeats within the genome, MLVA enables high-resolution differentiation of strains, and offering valuable insights into the genetic makeup of Brucella. 22 , 24 The faster accumulation of genetic variation in tandem repeat markers compared to SNP-based variation, allows genotyping and distinguishing between closely related Brucella strains. 25 MLVA technique is cost-effective and serves as an excellent alternative to other sequencing methods. 22 Due to the zoonotic nature of brucellosis, tracing infection sources, identifying transmission pathways, and implementing effective control strategies are crucial for epidemiologic surveillance in Oman. Accurately distinguishing between Brucella species and biovars is important for treatment, controlling the disease, and developing of a common vaccine. DNA-based genotyping methods are preferred, as they offer greater discriminatory power compared to phenotypic methods. This study aimed to investigate the genetic makeup of Brucella strains circulating in different geographical locations in Oman in both human and livestock using MLVA. Methods Ethical considerations This study was approved by the Medical Research Ethics Committee (MERC) in the College of Medicine & Health Sciences at Sultan Qaboos University (SQU) REF. NO. SQU-EC/060/2023 for using human samples. The research project submitted to the Medical Research Ethics Committee (MREC), College of Medicine and Health Sciences, Sultan Qaboos University for re-consideration and approval was discussed. The modifications received by the Committee on 3rd May 2023 in response to the comments raised during the 30th March 2023 meeting were found to be satisfactory. The Committee has considered the research project acceptable and therefore approval was granted. For animal samples, there was no need for the Sultan Qaboos University ethical committee’s approval as the study did not involve experimental research on animals. The samples were used after verbal consent from the patients without disclosing their names. The animals were needed only for milk samples. The milking was done in a clean quiet area to ensure a peaceful environment for the animals. There was minimal handling and restraint under the consent, help, and guidance of the owners who are very familiar with their stock. Milking was carried out aseptically by experienced technicians. The clinician was around to ensure the safety of the animals during the procedure. This asserts that animal welfare was not compromised in any way more than minimal normal handling for aseptic milking under clinical supervision in the presence of animal owners. One hundred and sixteen milk samples from different animal species and twenty-one human blood samples were collected from Dhofar and AD Dakhiliyah governorates in Oman. Brucella in human blood samples was identified by incubating blood in an aerobic blood medium vial. Then, samples were incubated in the BD BACTEC™ blood culture system. After that, the samples were streaked directly into the blood and chocolate agar media simultaneously and incubated for 24-48 hours. Gram staining and biochemical tests (urease and oxidase) were performed for confirmation. Milk samples were screened for Brucella presence using a milk ring and rapid tests. Then, seropositive samples were incubated for 24 hours at 37°C in a shaker incubator. Subsequently, samples were centrifuged, the supernatant was removed, and 200 μL of phosphate buffer saline (PBS) was added to the pellet. DNA extraction was done for all samples as previously described by Yu & Morrison. 26 To confirm the presence of Brucella genome in milk samples, real-time PCR was carried out using the BruSpp dtec-qPCR kit (Genetic Analysis Strategies, Spain) followed by conventional PCR based on three Brucella -specific primer pairs as previously described. 27 – 29 Moreover, species-specific primers (Bruce-ladder) were employed, as previously described. 21 MLVA genotyping of B. melitensis was carried out using the BRUCELLA MLVA-16 Typing Kit (Genoscreen, France), where 16 markers are amplified from purified DNA using four quadruplex PCR reactions; the refined set of 16 VNTR was previously outlined. 7 , 30 MLVA-16 analysis was performed on 21 human isolates, a goat isolate, and 42 DNA extracted from milk samples. Specifically for milk samples, eight markers (Bruce 04, 11, 45, 30, 21, 43, 42, and 19) were amplified through singleplex PCR. Each PCR was conducted under identical parameters to ensure consistency across samples. The PCR products from the singleplex reactions were pooled into two multiplex groups before undergoing capillary electrophoresis. Capillary electrophoresis and fragment analysis of PCR products were conducted by two companies: GenoScreen (France) and Macrogen (South Korea). All PCR products were run on an ABI 3730XL capillary sequencer 44 utilizing POP7 polymer to ensure high-resolution fragment separation. Two size standards were applied based on the service provider. GeneScan 600LIZ size standard (Applied Biosystems) was used for samples analyzed by macrogen. GeneScan 1200LIZ size standard (Applied Biosystems) was used for samples analyzed by GenoScreen. Data analysis Data of Brucella strain genotypes of this study were compared with MLVA genotypes of the Brucella MLVA website ( http://mlva.u-psud.fr/brucella/ ) in November 2024. Band size estimates were translated into a number of units using BioNumerics version 7.6 (Applied Maths, Belgium). For clustering analyses (dendrogram), the categorical coefficient and the unweighted-pair group method using average linkages (UPGMA) algorithm were used. Minimum Spanning Trees (MST) were constructed using BioNumerics applying categorical coefficients together with the single and double locus variance priority rules. Results Peaks corresponding to all 16 markers were detected in all isolates (human and goat) by capillary electrophoresis. However, only 12 out of 42 milk samples showed peaks for all 16 markers. The remaining samples lacked at least one locus. Therefore, MLVA-14 was selected as a typing approach since it allowed the inclusion of samples missing up to two loci, while excluding those missing three or more loci. As a result, 27/42 milk samples were amplified for all 14 markers (Bruce8, Bruce11, Bruce43, Bruce45, Bruce21, Bruce6, Bruce42, Bruce18, Bruce4, Bruce9, Bruce16, Bruce30, Bruce19). No sample showed multiple alleles at any locus. MLVA-14 genotyping of 49 B. melitensis strains showed that Bruce8, Bruce11, Bruce43, Bruce45 and Bruce21 loci were homogenous. Conversely, the most discriminatory loci were Bruce6, Bruce42, Bruce18, Bruce4, Bruce9, Bruce16, Bruce30 and Bruce19 ( Table 1 ). Subsequent cluster analysis allowed to recognize two main B. melitensis clusters including 31 different MLVA-14 genotypes ( Figure 1 ). The distances between strains within and between clusters were calculated based on the number of matching and differing VNTRs. In Dhofar governorate, B. melitensis strains extracted from camel milk (K187, K189, K185, K188) and goat milk (K197) in 2023 shared the same genotype. Moreover, B. melitensis strains extracted from goat milk (K43, K44, K47, K48, K50) had the same genotype as strains recovered from cattle milk (K55, K56, K60). In addition, strains retrieved from camel milk (K102, K103, K104, K107) shared the same genotype as strains from cattle milk (K112, K113). On the other hand, two human isolates (K219, K220) dating 2024 had the same genotype; also, the other two human isolates (K212, K216), identified in the same year, shared a unique genotype. The rest of the strains showed different genotypes thus forming subclusters ( Figure 1 ). Table 1. Genetic diversity of 49 B. melitensis strains based on MLVA-14 genotyping. Locus Number Number of alleles per locus Number of copies of tandem repeats in locus Bruce 30 4 4, 5, 6, 7 Bruce 19 2 36, 41 Bruce 42 3 2, 3, 4 Bruce 18 4 3, 4, 5, 7 Bruce 43 1 2 Bruce 21 1 8 Bruce 09 7 2, 3, 4, 5, 6, 7, 8 Bruce 12 2 12, 13 Bruce 06 3 1, 2, 3 Bruce 08 1 5 Bruce 16 5 4, 5, 6, 8, 10 Bruce 04 7 2, 3, 4, 5, 6, 7, 8 Bruce 45 1 3 Bruce 11 1 3 Figure 1. Dendrogram based on MLVA-14 genotyping using UPGMA (Unweighted Pair Group Method with Arithmetic Mean). The figure shows the genetic relationship among 49 B. melitensis strains recovered from human and different animal species in Oman. The columns represent the MLVA-14 profile, key (identification number), sample type, collection year, species, and geographic origin respectively. Minimum spanning trees (MST) were generated for the 49 Omani strains based on host species and geographical location ( Figure 2 ). MST presents the genetic profiles, with circles representing individual or grouped strains and edges reflecting genetic distances. Circle colors indicate the host; green (human), red (goat), purple (camel), and yellow (cattle), while circle sizes correspond to the number of isolates sharing an identical genetic profile. Human isolates (green) were related, suggesting a shared genetic background. Brucella strains extracted from goat milk (red) showed distinct clustering, with some overlap with strains extracted from camel milk (purple) and cattle milk (yellow). For geographic annotations, most strains originated from Dhofar Governorate, two human isolates from AD Dakheliya Governorate and a goat isolate from Al Jabal Al Akhdhar which located in AD Dakheliya Governorate. The phylogeographic patterns of the studied Brucella strains were compared to MLVA profiles available in the international database ( Figure 3 & Figure 4 ). It should be noted that for MST worldwide analysis, Bruce19 locus was not included since many foreign isolates were not typed for this marker and to avoid numbering uncertainty due to the recent discovery of rare alleles at the Bruce19 locus that changed the nomenclature. 11 The Omani strains belong to the East Mediterranean lineage. In the MST analysis with worldwide isolates, most Omani strains tend to separate forming a country branch, which includes two strains from the United Arab Emirates (in brown). However, another group of Oman strains clusters with Spain, Portugal, India, Turkey, and China strains. These two Omani clusters are connected to a junction node that includes isolates from Portugal, China, France and Kazakhstan. Finally, it can be noticed one “outlier” Oman strain correlated with a big cluster mostly formed by isolates from China, but also including strains from Portugal, Kazakhstan, Turkey, Mongolia, Marocco, France and Spain. Figure 2. MLVA-14 Minimum Spanning Tree describing the relationships of 49 B. melitensis strains based on species and location. Circles represent MLVA-14 genotypes, colored according to the species, and the size of the circle indicates the number of strains within that genotype . Figure 3. MLVA-14 Minimum Spanning Tree describing the relationships of 49 B. melitensis isolates with worldwide isolates. Circles represent MLVA-14 genotypes, colored according to the country of origin, and the size of the circle indicates the number of strains within that genotype. Figure 4. MLVA-14 Minimum Spanning Tree (zoomed) describing the relationships of 49 B. melitensis isolates with worldwide isolates with strains IDs. Circles represent MLVA-14 genotypes, colored according to the country of origin, and the size of the circle indicates the number of strains within that genotype. Discussion This study aimed to genotype Brucella strains circulating in human and livestock in Oman. To confirm the presence of Brucella in milk samples, a milk ring and rapid tests were used. Then, real-time PCR was also carried out, followed by conventional PCR based on three Brucella -specific primer pairs. Moreover, species-specific primers (Bruce-ladder) were employed, as described by Ref. 21 . This study, to the best of our knowledge, is the first to use DNA extracted directly from milk samples for MLVA analysis. This approach likely explains the missing loci in the PCR products of some samples. Moreover, extraction of DNA directly from milk may yield low-quality DNA. Also, the presence of PCR inhibitors in milk may hinder PCR reactions. In general, it should be taken into consideration that milk samples might contain more than one Brucella isolates, and multiple alleles can be amplified at certain loci thus hampering inference of MLVA haplotypes. Such occurrence seems to be unlikely given the fact that, in this study, all milk samples investigated were infected by a single strain; however, the possibility to detect co-infections with multiple strains would obviously increase when working with bulk milk samples in endemic areas. Nonetheless, we decided to work with milk samples to avoid the risk associated with culturing Brucella. Moreover, the lack of a biosafety level 3 cabinet (BSL-3) at the microbiology laboratory in the animal and veterinary sciences department in SQU was an obstacle to dealing with the zoonotic nature of Brucella. It appears that it is worth mentioning that all human isolates used in this study were cultured at Sultan Qaboos University Hospital (SQUH) and Sultan Qaboos Hospital in Salalah (SQH). All these isolates were successfully typed at each locus for the 16 markers. Therefore, isolates remain the best choice for MLVA analysis. Interestingly, even a study by Ref. 31 using isolates had reported missing loci for some markers, such as Bruce07 and Bruce19, underscoring potential challenges in MLVA analysis. Consequently, MLVA-14 analysis was utilized to genotype 49 B. melitensis strains, clustered into two main groups comprising 31 distinct genotypes. Based on the dendrogram ( Figure 1 ) and ( Table 1 ), homogeneity was observed in Bruce8, Bruce11, Bruce43, Bruce45, and Bruce21 loci with a monomorphic profile in other studies. 7 , 32 On the other hand, Bruce 21 presented variable allelic types in previous studies. 7 , 11 , 31 , 33 In this study, Bruce6, Bruce42, Bruce12, and Bruce19 loci were moderately variable with two to three allelic types, also moderately variable in other studies. 11 , 30 The highly discriminatory markers in this study were found in Bruce4, Bruce9, Bruce16, Bruce18, and Bruce30. Particularly, Bruce4 and Bruce9 exhibited diverse allelic types (7 types), contributing significantly to the differentiation between Brucella strains. In a study by Tiller et al. (2019), Bruce4, Bruce9, Bruce16, and Bruce18 were also polymorphic, whereas Bruce 30 tended to be more conserved. 32 Kulakov et al. (2011) reported that Bruce4 and Bruce16 were highly polymorphic, whereas Bruce9 and Bruce18 were monomorphic. 34 In another study, Bruce4, Bruce30, and Bruce16 were discriminatory markers. 11 Overall, the clustering patterns and genotypic differences or similarities observed in this study align with findings from previous research, emphasizing the usefulness of MLVA in studying Brucella genetic diversity and epidemiology. The genetic similarity observed among B. melitensis strains from camel milk (K187, K189, K185, K188) and goat milk (K197) samples in Dhofar Governorate suggests potential shared reservoirs or transmission pathways within livestock populations in the region. Similarly, the identical genotypes identified in goat milk strains (K43, K44, K47, K48, K50) and cattle milk strains (K55, K56, K60) highlight the interconnected nature of Brucella transmission across different animal hosts, possibly through shared environments, grazing lands, or interspecies interactions. Furthermore, the shared genotypes between camel milk strains (K102, K103, K104, K107) and cattle milk strains (K112, K113) reinforce the role of camels and cattle as significant reservoirs for B. melitensis in Dhofar Governorate. The transmission of B. melitensis between different animal species was reported previously in Oman. 4 , 5 , 35 – 37 Moreover, human isolates K219 and K220 shared the same genotype, while K212 and K216 formed another group with identical genotypes. These findings are consistent with previous studies which indicate that human infection is often linked to livestock reservoirs due to zoonotic transmission, frequently through the consumption of unpasteurized dairy products or direct contact with infected animals. 36 , 38 – 41 In addition, the majority of human cases in this study reported a history of consuming unpasteurized raw milk across all age groups according to SQUH and SQH. Notably, the highest number of cases was observed among children under the age of ten years. However, that might be because of their developing immune systems, making them more susceptible to infections like Brucella. Additionally, children are more likely to consume unpasteurized milk, especially in regions where it is considered a traditional dietary essential. Limited awareness of the risks associated with raw milk consumption, combined with potential exposure to contaminated environments, such as farms or infected animals, further increases their risk. Advancement of the healthcare system in Oman has led to improved diagnosis and reporting of illnesses across all age groups. The genetic heterogeneity of other strains, divided into subclusters, reflects the diverse Brucella population in Dhofar Governorate, highlighting the need for localized control measures and management procedures. 4 , 36 , 37 Our MLVA analysis provides valuable insights also into the epidemiology of Brucella in Oman, including host specificity, geographic clustering, and potential transmission dynamics. The phylogeographic analysis of 49 Brucella melitensis strains from Oman was compared to international MLVA-15 profiles as previously described. 7 , 30 , 33 This provides significant insights into the genetic relationships and potential transmission patterns of these strains worldwide. In MST analysis integrating worldwide isolates, most Omani strains formed a distinct country-specific branch, with close association with two strains from the United Arab Emirates. Specifically, this branch included strains recovered from human (K234, K239, K244), cattle and camel milk (K53, K70, K102, K103, K107, K104, K112, K113). This finding suggests a localized genetic lineage, likely shaped by regional factors such as shared livestock trade routes or common environmental reservoirs in the Arabian Peninsula. 4 , 42 Additionally, another cluster of Omani strains recovered from goat and cattle milk (K143, K144, K147, K148, k150, K155, K156, K160) grouped with isolates from Spain, Portugal, India, Turkey, and China. This broader clustering indicates potential historical or trade-driven links, reflecting the global trade of livestock or animal products, which has likely influenced the genetic diversity of B. melitensis strains. 35 , 36 Interestingly, one “outlier” Omani goat isolate (K179) was closely related to a cluster including mainly Chinese isolates, but also isolates from countries worldwide (i.e., Portugal, Kazakhstan, Mongolia, Turkey, Spain, Morocco, France) highlighting an exception to the broader clustering trends. This unique pattern may represent a rare introduction of a strain through trade, livestock importation, and, or human travel. It appears that the complexity of the trading economy may explain some of the observed genetic relationships; for example, Oman imports dairy products, eggs, honey, and other edible products from Kazakhstan. These results underscore the complexity of Brucella phylogeography, reflecting both regional and global transmission dynamics. The distinct clustering of most Omani strains emphasizes the potential role of localized environmental and epidemiological factors in shaping Brucella diversity. However, the observed connections with strains from other countries emphasize the importance of monitoring international trade and the movement of livestock to prevent the spreading of brucellosis. 43 Conclusions This research is the first to genotype, compare and correlate B. melitensis strains from humans and livestock in Oman, through MLVA-14. The results illustrate a genetic relationship between strains of human and livestock origin, further illustrating zoonotic transmission and making these populations interconnected. Also, the association of the genotypes and their distribution among different animal species corroborates the established transmission routes among livestock in Dhofar and AD Dakhiliya Governorates. Isolates produced relatively better genotyping than milk samples, thus demonstrating the need for careful and proper sample selection and handling. Building on these results, however, there is a need for a One Health approach involving both the veterinary and the public health sectors in brucellosis control efforts. Major next steps include broadening the sampling base from other governorates in Oman, implementing whole genome sequencing together with MLVA for even more comparative studies, and developing a national Brucella surveillance strategy that incorporates a centralized genotype repository. Finally, strengthening laboratory capacities through expertise and equipment for Brucella isolation and characterization would improve the surveillance system and disease management in Oman. This study was approved by the Medical Research Ethics Committee (MERC) in the College of Medicine & Health Sciences at Sultan Qaboos University (SQU) REF. NO. SQU-EC/060/2023 for using human samples. The research project submitted to the Medical Research Ethics Committee (MREC), College of Medicine and Health Sciences, Sultan Qaboos University for re-consideration and approval was discussed. The modifications received by the Committee on 3rd May 2023 in response to the comments raise during the 30th March 2023 meeting were found to be satisfactory. The Committee has considered the research project acceptable and therefore approval was granted. For animal samples, there was no need for the Sultan Qaboos University ethical committee’s approval as the study did not involve experimental research on animals. The animal milk samples were submitted to the central laboratory for routine diagnosis and a verbal consent was granted to use the samples for our project. Consent to participants All participants gave an oral consent to participate in this research work. Animal ethics The experiment was conducted in Dhofar and AD Dakhiliyah governorates, Oman, for milk samples. The samples were collected from grazing animals in the open, owned by private individuals with their consent and guidance, who managed animal husbandry as per rural tradition. There was minimal interaction with the animals for the collection of milk for further investigation. Minimal animal manipulation (handling & restraint) was observed for milking by experienced personnel, ensuring an aseptic collection procedure. The sample size was much smaller than the normal milking volume for each animal. The animals were needed only for milk samples. The milking was done in a clean quiet area to ensure a peaceful environment for the animals. There was minimal handling and restraint under the consent, help, and guidance of the owners who are very familiar with their stock. Milking was carried out aseptically by experienced technicians. The clinician was around to ensure the safety of the animals during the procedure. This asserts that animal welfare was not compromised in any way more than minimal normal handling for aseptic milking under clinical supervision in the presence of animal owners. Data availability statement Figshare: Genetic diversity of brucella melitensis in Oman, https://doi.org/10.6084/m9.figshare.28190633 . 44 Title: Paper-Raw Data.zip Author: Yasmin Ahmed Description: Genetic diversity of brucella melitensis in Oman It contains three files 1. FSA files contain the Capillary electrophoresis raw data 2. An excel file contains comparison between Oman strains & worldwide strains 3. A word document file with detailed description of human & animal samples used in the study. Names of human patients were not revealed. Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0). Acknowledgements Thanks go to Sultan Qaboos University Hospital specifically Suhaim Al-Ghafri and Sultan Qaboos Hospital in Salalah especially Abir Ba Hajjaj for cooperation and providing the human samples. Also, our gratitude extends to the Central Animal Health Laboratory, particularly Mr. Taha Al-Subhi, for supplying the animal samples. References 1. Georgios P, Photini P, Nikolaos A, et al. : The New Global Map of Human Brucellosis. The new global map of human brucellosis. 2006. 2. 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Moreno E, Blasco JM, Moriyón I: Facing the Human and Animal Brucellosis Conundrums: The Forgotten Lessons. Microorganisms. 2022; 10 (5). PubMed Abstract | Publisher Full Text | Free Full Text 44. Yasmin Ahmed paper-Raw Data. Zip. [Dataset]. 2024. license. Publisher Full Text Reference Source Comments on this article Comments (0) Version 2 VERSION 2 PUBLISHED 12 Feb 2025 ADD YOUR COMMENT Comment Author details Author details 1 Central Laboratory for Animal Health, Department of Animal Diseases Diagnosis, Oman, Muscat, Oman 2 Animal & Veterinary Sciences, Sultan Qaboos University, Muscat, Muscat Governorate, Oman 3 Microbiology & Immunology, Sultan Qaboos University, Muscat, Muscat Governorate, Oman 4 Biological Safety, Kazakh National Agrarian Research University, Almaty, Kazakhstan 5 Istituto Zooprofilattico Sperimentale del Piemonte, Turin, Italy Khalsa Altoubi Roles: Investigation, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing Zakariya Al Muharrmi Roles: Investigation, Methodology, Supervision Waleed AlMarzooqi Roles: Investigation, Methodology, Supervision Salma Al Adwani Roles: Supervision, Writing – Review & Editing Kaadhia Al Kharousi Roles: Investigation, Methodology Shytyrbayeva Zamzagul Abdildaevna Roles: Software, Writing – Review & Editing Simone Peletto Roles: Investigation, Methodology, Writing – Review & Editing Yasmin ElTahir Roles: Investigation, Methodology, Project Administration, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing Competing interests No competing interests were disclosed. Grant information This study was supported by the research council (RC/RG-AGR/ANVS/21/01). The funders had no role in the design of the study, data collection, analyses, decision to publish or preparation of the manuscript. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Article Versions (2) version 2 Revised Published: 13 Mar 2026, 14:191 https://doi.org/10.12688/f1000research.161111.2 version 1 Published: 12 Feb 2025, 14:191 https://doi.org/10.12688/f1000research.161111.1 Copyright © 2025 Altoubi K et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Download Export To Sciwheel Bibtex EndNote ProCite Ref. Manager (RIS) Sente metrics Views Downloads F1000Research - - PubMed Central info_outline Data from PMC are received and updated monthly. - - Citations open_in_new 0 open_in_new 0 open_in_new SEE MORE DETAILS CITE how to cite this article Altoubi K, Al Muharrmi Z, AlMarzooqi W et al. East Mediterranean Lineage of Brucella melitensis in Human Isolates and Milk Samples in Oman Using MLVA-14 [version 1; peer review: 1 approved with reservations] . F1000Research 2025, 14 :191 ( https://doi.org/10.12688/f1000research.161111.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS track receive updates on this article Track an article to receive email alerts on any updates to this article. TRACK THIS ARTICLE Share Open Peer Review Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Version 1 VERSION 1 PUBLISHED 12 Feb 2025 Views 0 Cite How to cite this report: Islam MS. Reviewer Report For: East Mediterranean Lineage of Brucella melitensis in Human Isolates and Milk Samples in Oman Using MLVA-14 [version 1; peer review: 1 approved with reservations] . F1000Research 2025, 14 :191 ( https://doi.org/10.5256/f1000research.177103.r388868 ) The direct URL for this report is: https://f1000research.com/articles/14-191/v1#referee-response-388868 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 14 Jun 2025 Md. Sadequl Islam , Hajee Mohammad Danesh Science and Technology University, Dinajpur, Bangladesh Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.177103.r388868 Introduction Critique: Clarity and Focus: The introduction provides a broad overview of brucellosis and its zoonotic importance. However, it lacks a sharply focused research gap or hypothesis. The shift from general background to specific ... Continue reading READ ALL Introduction Critique: Clarity and Focus: The introduction provides a broad overview of brucellosis and its zoonotic importance. However, it lacks a sharply focused research gap or hypothesis. The shift from general background to specific study objectives is gradual and could be more clearly demarcated. Redundancy: Multiple references to Brucella species and diagnostic tools make the narrative somewhat repetitive (e.g., PCR methods and serological tests are listed in detail without always linking back to the rationale for MLVA-14). Overload of Technical Details: The excessive explanation of general diagnostic methods (e.g., Rose Bengal, ELISA) dilutes the focus from the genetic diversity angle, which is the paper’s strength. Suggestions for Improvement: Explicitly highlight the research gap early (e.g., “No previous studies in Oman have analyzed the genetic diversity of B. melitensis using MLVA in both human and milk samples”). Trim redundant or overly detailed background information not directly tied to the study’s rationale. Consider ending the introduction with a concise, hypothesis-driven objective statement. Methods Critique: Strength: Well-detailed methodology, especially ethical approvals and sample handling—commendable from a biosafety and animal ethics perspective. Lack of Clarity in MLVA Details: Though the MLVA-16 and MLVA-14 transition is mentioned, the justification for reducing the number of markers (due to missing loci) could be clarified earlier. Reproducibility Gap: DNA quality control metrics and reasons for PCR failure in some samples could have been explained more robustly. Suggestions for Improvement: Include DNA quality thresholds or quantification methods. Add a flow diagram summarizing the sample collection, screening, and selection for final MLVA analysis. Results Suggestions for Improvement: Include a map showing sampling locations for geographic context. The study presents valuable MLVA-14 genotyping data on Brucella melitensis strains from Oman, contributing to the regional and global understanding of Brucella epidemiology. However, the manuscript does not indicate whether these genotypes have been submitted to the international MLVA Bank ( http://mlva.u-psud.fr/brucella/ ). I strongly recommend that the authors deposit the MLVA profiles in this publicly accessible database. Doing so will enhance the transparency, reproducibility, and comparative utility of the data for future studies and global surveillance efforts. Please include a statement confirming submission in the Methods or Data Availability section. Discussion Suggestions for Improvement: Consider including confidence metrics (e.g., bootstrap values) for MST branches to strengthen phylogeographic conclusions. Reframe speculative claims as hypotheses needing further exploration unless supported by trade/import data. Discuss limitations such as potential bias in sample collection (mostly Dhofar) and the use of milk vs isolate DNA. After the sentence in the discussion section please add the citation below “Overall, the clustering patterns and genotypic differences or similarities observed in this study align with findings from previous research, emphasizing the usefulness of MLVA in studying Brucella genetic diversity and epidemiology.” “Overall, the clustering patterns and genotypic differences or similarities observed in this study align with findings from previous research, emphasizing the usefulness of MLVA in studying Brucella genetic diversity and epidemiology. A similar molecular study conducted in Bangladesh reported the first genetic characterization of Brucella abortus biovar 3 using MLVA, reinforcing the value of this method in endemic regions for epidemiological insight and disease control planning (Islam et al., 2019).” Citation: Islam, M. S., Garofolo, G., Sacchini, L., Dainty, A. C., Khatun, M. M., Saha, S., & Islam, M. A. (2019). First isolation, identification, and genetic characterization of Brucella abortus biovar 3 from dairy cattle in Bangladesh. Veterinary Medicine and Science, 5 (4), 556–562. (refer to 1 )This addition provides a strong, real-world example from a comparable endemic region. Conclusion Critique: Overstated Generalization: The assertion that this is the “first study” using milk DNA in MLVA analysis may require qualification or citation. Missing Specific Recommendations: The section could benefit from more actionable public health recommendations. Suggestions for Improvement: Suggest policy-level implementations (e.g., pasteurization campaigns, livestock vaccination). Recommend steps for improving laboratory infrastructure based on study constraints. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Not applicable Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes References 1. Islam, M. S., Garofolo, G., Sacchini, L., Dainty, A. C., Khatun, M. M., Saha, S., & Islam, M. A. (2019). First isolation, identification, and genetic characterization of Brucella abortus biovar 3 from dairy cattle in Bangladesh. Veterinary Medicine and Science, 5(4), 556–562. https://doi.org/10.1002/vms3.511. 2. Ali E, Islam M, Hossen M, Khatun M, et al.: Extract of neem (Azadirachta indica ) leaf exhibits bactericidal effect against multidrug resistant pathogenic bacteria of poultry. Veterinary Medicine and Science . 2021; 7 (5): 1921-1927 Publisher Full Text Competing Interests: No competing interests were disclosed. Reviewer Expertise: Microbiology, Molecular biology, Enviromental toxicology, Gene expression I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Islam MS. Reviewer Report For: East Mediterranean Lineage of Brucella melitensis in Human Isolates and Milk Samples in Oman Using MLVA-14 [version 1; peer review: 1 approved with reservations] . F1000Research 2025, 14 :191 ( https://doi.org/10.5256/f1000research.177103.r388868 ) The direct URL for this report is: https://f1000research.com/articles/14-191/v1#referee-response-388868 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Comments on this article Comments (0) Version 2 VERSION 2 PUBLISHED 12 Feb 2025 ADD YOUR COMMENT Comment keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Reviewer Reports Invited Reviewers 1 2 Version 2 (revision) 13 Mar 26 read Version 1 12 Feb 25 read Md. Sadequl Islam , Hajee Mohammad Danesh Science and Technology University, Dinajpur, Bangladesh AbdulHamid Settenda Lukambagire , Kilimanjaro Clinical Research Institute, Moshi, Tanzania Comments on this article All Comments (0) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2026 Lukambagire A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 10 Apr 2026 | for Version 2 AbdulHamid Settenda Lukambagire , Kilimanjaro Clinical Research Institute, Moshi, Tanzania 0 Views copyright © 2026 Lukambagire A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions I feel there is too much detail in the ethical considerations, for instance this statement is unnecessary ... "The modifications received by the Committee on 3rd May 2023 in response to the comments raised during the 30th March 2023 meeting were found to be satisfactory. The Committee has considered the research project acceptable and therefore approval was granted." For Fig 1., although this diagram is rather informative, I would propose that the authors redesign it as a flow diagram/ step-wise diagram. It is rather difficult to follow the source, tests and samples highlighted therein. Also, since the authors have elected to include all the series of tests conducted on samples in this figure, it might be useful to also to provide a table/ summary of the various sample types and results (even as supplementary material). Also, how did the authors account for the potential low DNA yield from direct milk extraction to allow MLVA typing? A map to show the relative locations of the sample sources would be useful for readers not familiar with the geography of the country or region in general. I found the discussion points quite well detailed and compared with previous work. However, I found it very difficult to distill the point each of these paragraphs was trying to put across. In the first paragraph of the discussion, authors mention multiple points, most of which actually sound like limitations to the study findings/ inferences. I would propose these are clearly stated in a final paragraph before conclusions. The second paragraph also discusses a lot about genotypes and loci...but doesn't clearly communicate a take home message from this?! I would presume that two core clusters (with various loci) were observed...one fairly conserved, as observed in previous studies, and another quite variable. All these points are critical to the overall understanding of the work done, but I feel the authors have left these deductions to the discernment of the reader, which is rather self-defeating. It all leads to a conclusion that is not very well appreciated given the great study design and results presented. I would recommend the discussion section is re-written to provide succinct take-home language. Lastly, there is a lot of repeated ethical language abutted to the conclusion paragraph. Since most of this language is repeated from the methods section, this should either be removed completely, or rewritten and summarized under an ethics statement (review journal format for this). Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise Brucella (zoonotic disease) diagnosis, molecular epidemiology, immunology; Medical parasitology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (0) Lukambagire AS. Peer Review Report For: East Mediterranean Lineage of Brucella melitensis in Human Isolates and Milk Samples in Oman Using MLVA-14 [version 1; peer review: 1 approved with reservations] . F1000Research 2025, 14 :191 ( https://doi.org/10.5256/f1000research.196492.r469956) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-191/v2#referee-response-469956 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Islam M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 14 Jun 2025 | for Version 1 Md. Sadequl Islam , Hajee Mohammad Danesh Science and Technology University, Dinajpur, Bangladesh 0 Views copyright © 2025 Islam M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Introduction Critique: Clarity and Focus: The introduction provides a broad overview of brucellosis and its zoonotic importance. However, it lacks a sharply focused research gap or hypothesis. The shift from general background to specific study objectives is gradual and could be more clearly demarcated. Redundancy: Multiple references to Brucella species and diagnostic tools make the narrative somewhat repetitive (e.g., PCR methods and serological tests are listed in detail without always linking back to the rationale for MLVA-14). Overload of Technical Details: The excessive explanation of general diagnostic methods (e.g., Rose Bengal, ELISA) dilutes the focus from the genetic diversity angle, which is the paper’s strength. Suggestions for Improvement: Explicitly highlight the research gap early (e.g., “No previous studies in Oman have analyzed the genetic diversity of B. melitensis using MLVA in both human and milk samples”). Trim redundant or overly detailed background information not directly tied to the study’s rationale. Consider ending the introduction with a concise, hypothesis-driven objective statement. Methods Critique: Strength: Well-detailed methodology, especially ethical approvals and sample handling—commendable from a biosafety and animal ethics perspective. Lack of Clarity in MLVA Details: Though the MLVA-16 and MLVA-14 transition is mentioned, the justification for reducing the number of markers (due to missing loci) could be clarified earlier. Reproducibility Gap: DNA quality control metrics and reasons for PCR failure in some samples could have been explained more robustly. Suggestions for Improvement: Include DNA quality thresholds or quantification methods. Add a flow diagram summarizing the sample collection, screening, and selection for final MLVA analysis. Results Suggestions for Improvement: Include a map showing sampling locations for geographic context. The study presents valuable MLVA-14 genotyping data on Brucella melitensis strains from Oman, contributing to the regional and global understanding of Brucella epidemiology. However, the manuscript does not indicate whether these genotypes have been submitted to the international MLVA Bank ( http://mlva.u-psud.fr/brucella/ ). I strongly recommend that the authors deposit the MLVA profiles in this publicly accessible database. Doing so will enhance the transparency, reproducibility, and comparative utility of the data for future studies and global surveillance efforts. Please include a statement confirming submission in the Methods or Data Availability section. Discussion Suggestions for Improvement: Consider including confidence metrics (e.g., bootstrap values) for MST branches to strengthen phylogeographic conclusions. Reframe speculative claims as hypotheses needing further exploration unless supported by trade/import data. Discuss limitations such as potential bias in sample collection (mostly Dhofar) and the use of milk vs isolate DNA. After the sentence in the discussion section please add the citation below “Overall, the clustering patterns and genotypic differences or similarities observed in this study align with findings from previous research, emphasizing the usefulness of MLVA in studying Brucella genetic diversity and epidemiology.” “Overall, the clustering patterns and genotypic differences or similarities observed in this study align with findings from previous research, emphasizing the usefulness of MLVA in studying Brucella genetic diversity and epidemiology. A similar molecular study conducted in Bangladesh reported the first genetic characterization of Brucella abortus biovar 3 using MLVA, reinforcing the value of this method in endemic regions for epidemiological insight and disease control planning (Islam et al., 2019).” Citation: Islam, M. S., Garofolo, G., Sacchini, L., Dainty, A. C., Khatun, M. M., Saha, S., & Islam, M. A. (2019). First isolation, identification, and genetic characterization of Brucella abortus biovar 3 from dairy cattle in Bangladesh. Veterinary Medicine and Science, 5 (4), 556–562. (refer to 1 )This addition provides a strong, real-world example from a comparable endemic region. Conclusion Critique: Overstated Generalization: The assertion that this is the “first study” using milk DNA in MLVA analysis may require qualification or citation. Missing Specific Recommendations: The section could benefit from more actionable public health recommendations. Suggestions for Improvement: Suggest policy-level implementations (e.g., pasteurization campaigns, livestock vaccination). Recommend steps for improving laboratory infrastructure based on study constraints. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Not applicable Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes References 1. Islam, M. S., Garofolo, G., Sacchini, L., Dainty, A. C., Khatun, M. M., Saha, S., & Islam, M. A. (2019). First isolation, identification, and genetic characterization of Brucella abortus biovar 3 from dairy cattle in Bangladesh. Veterinary Medicine and Science, 5(4), 556–562. https://doi.org/10.1002/vms3.511. 2. Ali E, Islam M, Hossen M, Khatun M, et al.: Extract of neem (Azadirachta indica ) leaf exhibits bactericidal effect against multidrug resistant pathogenic bacteria of poultry. Veterinary Medicine and Science . 2021; 7 (5): 1921-1927 Publisher Full Text Competing Interests No competing interests were disclosed. Reviewer Expertise Microbiology, Molecular biology, Enviromental toxicology, Gene expression I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (0) Islam MS. Peer Review Report For: East Mediterranean Lineage of Brucella melitensis in Human Isolates and Milk Samples in Oman Using MLVA-14 [version 1; peer review: 1 approved with reservations] . F1000Research 2025, 14 :191 ( https://doi.org/10.5256/f1000research.177103.r388868) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. 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