PacBio sequencing revealed variation in the microbiota diversity, species richness and composition between milk collected from healthy and mastitis cows

Ma, Teng; Shen, Lingling; Wen, Qiannan; Lv, Ruirui; Hou, Qiangchuan; Kwok, Lai Yu; Sun, Zhihong; Zhang, Heping

doi:10.1099/mic.0.000968

Cited by 7 publications

(4 citation statements)

References 70 publications

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“…It is easy to survive and reproduce in a moist environment, which leads to cow mastitis [57,58]. Ma et al showed that there was a significant negative correlation between Acinetobacter_johnsonii and Lactococcus, and Lactococcus could inhibit the adhesion of common bovine mastitis pathogens to mammary gland cells and reduce the histological changes of mammary glands, so the antagonism of Acinetobacter_johnsonii and Lactococcus may indirectly aggravate the occurrence of mastitis [59,60]. Pseudomonas_aeruginosa is one of the pathogens of bovine mastitis.…”

Section: Discussionmentioning

confidence: 99%

A Study on Differential Biomarkers in the Milk of Holstein Cows with Different Somatic Cells Count Levels

She

Liu

et al. 2023

Animals

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Dairy cow mastitis is one of the common diseases of dairy cows, which will not only endanger the health of dairy cows but also affect the quality of milk. Dairy cow mastitis is an inflammatory reaction caused by pathogenic microorganisms and physical and chemical factors in dairy cow mammary glands. The number of SCC in the milk of dairy cows with different degrees of mastitis will increase in varying degrees. The rapid diagnosis of dairy cow mastitis is of great significance for dairy cow health and farm economy. Based on the results of many studies on the relationship between mastitis and somatic cell count in dairy cows, microflora, and metabolites in the milk of Holstein cows with low somatic cell level (SCC less than 200,000), medium somatic cell level (SCC up to 200,000 but less than 500,000) and high somatic cell level (SCC up to 5000,00) were analyzed by microbiome and metabolic group techniques. The results showed that there were significant differences in milk microbiota and metabolites among the three groups (p < 0.05), and there was a significant correlation between microbiota and metabolites. Meanwhile, in this experiment, 75 differential metabolites were identified in the H group and L group, 40 differential metabolites were identified in the M group and L group, and six differential microorganisms with LDA scores more than four were found in the H group and L group. These differential metabolites and differential microorganisms may become new biomarkers for the diagnosis, prevention, and treatment of cow mastitis in the future.

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Section: Discussionmentioning

confidence: 99%

A Study on Differential Biomarkers in the Milk of Holstein Cows with Different Somatic Cells Count Levels

She

Liu

et al. 2023

Animals

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“…Streptococcus originates primarily in the farm environment and is a common cause of dairy cow mastitis and most animals have no obvious clinical symptoms at the time of infection ( Moroni et al, 2006 ). S. suis has been also identified in raw milk in cows with mastitis ( Ma et al, 2021 ). We found S. suis in Hunan samples and this may be related to farm management practices.…”

Section: Discussionmentioning

confidence: 99%

Single Molecule Real-Time Sequencing and Traditional Cultivation Techniques Reveal Complex Community Structures and Regional Variations of Psychrotrophic Bacteria in Raw Milk

Meng

Liu

et al. 2022

Front. Microbiol.

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In this study, we investigated the species composition and diversity of psychrotrophic bacteria in raw milk from Heilongjiang, Inner Mongolia, Gansu, Henan, Anhui, Jiangsu, Chongqing, and Hunan provinces in China using traditional cultivation and PacBio Single Molecule Real-Time sequencing methods. The isolated psychrotrophic bacteria were highly diverse, which composed of 21 genera and 59 species. Pseudomonas accounted for 58.9% of the total genera while Stenotrophomonas and Enterococcus were also highly represented (above 5.0%). In particular, P. azotoformans occurred at a level of 16.9% and P. paralactis, P. lactis, E. faecalis, and P. marginalis were present in relatively high proportions (above 4.0%). Regional differences were found significantly among the test regions except samples from Heilongjiang and Inner Mongolia were similar. Additionally, differences were observed between days in Henan, Anhui, and Jiangsu samples. Therefore, control strategies must be implemented on regional and season basis.

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“…host cell/DNA exists. Most previous research that used PF (Ganda et al, 2016;Lima et al, 2018;Taponen et al, 2019;Al-Harbi et al, 2021;Ma T. et al, 2021;Schwenker et al, 2022) or NG kit (Quigley et al, 2012;Cheema et al, 2021;Lyons et al, 2021) considered 16S metagenomic sequencing for bacterial identification following DNA extraction; therefore, no host DNA contamination was reported. While amplicon sequencing is a sensitive method for taxonomy clarification, the relevance of this approach for rapid diagnostic and AMR gene detection in mastitis is questionable.…”

Section: Food-specific Bacterial Dna Isolation Kits Yielded a Lower N...mentioning

confidence: 99%

A culture-, amplification-independent, and rapid method for identification of pathogens and antibiotic resistance profile in bovine mastitis milk

et al. 2023

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IntroductionRapid and accurate diagnosis of causative pathogens in mastitis would minimize the imprudent use of antibiotics and, therefore, reduce the spread of antimicrobial resistance. Whole genome sequencing offers a unique opportunity to study the microbial community and antimicrobial resistance (AMR) in mastitis. However, the complexity of milk samples and the presence of a high amount of host DNA in milk from infected udders often make this very challenging.MethodsHere, we tested 24 bovine milk samples (18 mastitis and six non-mastitis) using four different commercial kits (Qiagens’ DNeasy® PowerFood® Microbial, Norgens’ Milk Bacterial DNA Isolation, and Molzyms’ MolYsis™ Plus and Complete5) in combination with filtration, low-speed centrifugation, nuclease, and 10% bile extract of male bovine (Ox bile). Isolated DNA was quantified, checked for the presence/absence of host and pathogen using PCR and sequenced using MinION nanopore sequencing. Bioinformatics analysis was performed for taxonomic classification and antimicrobial resistance gene detection.ResultsThe results showed that kits designed explicitly for bacterial DNA isolation from food and dairy matrices could not deplete/minimize host DNA. Following using MolYsis™ Complete 5 + 10% Ox bile + micrococcal nuclease combination, on average, 17% and 66.5% of reads were classified as bovine and Staphylococcus aureus reads, respectively. This combination also effectively enriched other mastitis pathogens, including Escherichia coli and Streptococcus dysgalactiae. Furthermore, using this approach, we identified important AMR genes such as Tet (A), Tet (38), fosB-Saur, and blaZ. We showed that even 40 min of the MinION run was enough for bacterial identification and detecting the first AMR gene.ConclusionWe implemented an effective method (sensitivity of 100% and specificity of 92.3%) for host DNA removal and bacterial DNA enrichment (both gram-negative and positive) directly from bovine mastitis milk. To the best of our knowledge, this is the first culture- and amplification-independent study using nanopore-based metagenomic sequencing for real-time detection of the pathogen (within 5 hours) and the AMR profile (within 5–9 hours), in mastitis milk samples. These results provide a promising and potential future on-farm adaptable approach for better clinical management of mastitis.

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PacBio sequencing revealed variation in the microbiota diversity, species richness and composition between milk collected from healthy and mastitis cows

Cited by 7 publications

References 70 publications

A Study on Differential Biomarkers in the Milk of Holstein Cows with Different Somatic Cells Count Levels

A Study on Differential Biomarkers in the Milk of Holstein Cows with Different Somatic Cells Count Levels

Single Molecule Real-Time Sequencing and Traditional Cultivation Techniques Reveal Complex Community Structures and Regional Variations of Psychrotrophic Bacteria in Raw Milk

A culture-, amplification-independent, and rapid method for identification of pathogens and antibiotic resistance profile in bovine mastitis milk

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