BACKGROUND: Acinetobacter lwoffii (A. lwoffii) is a Gram-negative bacteria common in the environment, and it is the normal flora in human respiratory and digestive tracts. The bacteria is a zoonotic and opportunistic pathogen that causes various infections, including nosocomial infections. The aim of this study was to identify A. lwoffii strains isolated from bovine milk with subclinical mastitis in China and get a better understanding of its antimicrobial susceptibility and resistance profile. This is the first study to analyze the drug resistance spectrum and corresponding mechanisms of A. lwoffii isolated in raw milk.
RESULTS: Four A. lwoffii strains were isolated by PCR method. Genetic evolution analysis using the neighbor-joining method showed that the four strains had a high homology with Acinetobacter lwoffii. The strains were resistant to several antibiotics and carried 17 drug-resistance genes across them. Specifically, among 23 antibiotics, the strains were completely susceptible to 6 antibiotics, including doxycycline, erythromycin, polymyxin, clindamycin, imipenem, and meropenem. In addition, the strains showed variable resistance patterns. A total of 17 resistance genes, including plasmid-mediated resistance genes, were detected across the four strains. These genes mediated resistance to 5 classes of antimicrobials, including beta-lactam, aminoglycosides, fluoroquinolones, tetracycline, sulfonamides, and chloramphenicol.
CONCLUSIONS: These findings indicated that multi-drug resistant Acinetobacter lwoffii strains exist in raw milk of bovine with subclinical mastitis. Acinetobacter lwoffii are widespread in natural environmental samples, including water, soil, bathtub, soap box, skin, pharynx, conjunctiva, saliva, gastrointestinal tract, and vaginal secretions. The strains carry resistance genes in mobile genetic elements to enhance the spread of these genes. Therefore, more attention should be paid to epidemiological surveillance and drug resistant A. lwoffii.

Subclinical mastitis is a common disease that threatens the welfare and health of dairy cows and causes huge economic losses. Somatic cell count (SCC) is the most suitable indirect index used to evaluate the degree of mastitis. To explore the relationship between SCC, diversity in the microbiome, and subclinical mastitis, we performed next-generation sequencing of the 16S rRNA gene of cow\'s milk with different SCC ranges. The data obtained showed that the microbiota was rich and coordinated with SCC below 2 × 105. SCC above 2 × 105 showed a decrease in the diversity of microbial genera. When SCC was below 2 × 105, the phylum Actinobacteriota accounted for the most. When SCC was between 2 × 105 and 5 × 105, Firmicutes accounted for the most, and when SCC exceeded 5 × 105, Firmicutes and Proteobacteria accounted for the most. Pathogenic genera such as Streptococcus spp. were absent, while SCC above 2 × 105 showed a decrease in the diversity of microbial genera. SCC was positively correlated with the percentage of Romboutsia, Turicibacter, and Paeniclostridium and negatively correlated with the percentage of Staphylococcus, Psychrobacter, Aerococcus, and Streptococcus. Romboutsia decreased 6.19 times after the SCC exceeded 2 × 105; the SCC increased exponentially from 2 × 105 to 5 × 105 and above 1 × 106 in Psychrobacter. Analysis of the microbiota of the different SCC ranges suggests that the development of mastitis may not only be a primary infection but may also be the result of dysbiosis in the mammary gland.

Teat cleaning pre- and post-milking is important for the overall health and hygiene of dairy cows. The purpose of this research was to evaluate the effectiveness of a teat detergents based on lactic acid bacteria according to changes in somatic cell count and cow-milk metabolites. Sixty-nine raw milk samples were collected from 11 Holstein-Friesian cows in China during 12 days of teat cleaning. An ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry-based untargeted metabolomic approach was applied to detect metabolomic differences after treatment with lactic acid bacteria and chemical teat detergents in cows with subclinical mastitis. The results suggest that the lactobacilli-based teat detergents could reduce somatic cell count and improve microhabitat of cow teat apex by adjusting the composition of metabolites. Furthermore, the somatic cell count could be decreased significantly within 10 days following the cleaning protocol. Lactic acid bacteria have the potential to be applied as a substitution to teat chemical detergents before and after milking for maintenance of healthy teats and breasts. Further, larger scale validation work is required to support the findings of the current study.

BACKGROUND: Folic acid is a water-soluble B vitamin (B9), which is closely related to the body\'s immune and other metabolic pathways. The folic acid synthesized by rumen microbes has been unable to meet the needs of high-yielding dairy cows. The incidence rate of subclinical mastitis in dairy herds worldwide ranged between 25%~65% with no obvious symptoms, but it significantly causes a decrease in lactation and milk quality. Therefore, this study aims at exploring the effects of folic acid supplementation on the expression profile of lncRNAs, exploring the molecular mechanism by which lncRNAs regulate immunity in subclinical mastitic dairy cows.
RESULTS: The analysis identified a total of 4384 lncRNA transcripts. Subsequently, differentially expressed lncRNAs in the comparison of two groups (SF vs. SC, HF vs. HC) were identified to be 84 and 55 respectively. Furthermore, the weighted gene co-expression network analysis (WGCNA) and the KEGG enrichment analysis result showed that folic acid supplementation affects inflammation and immune response-related pathways. The two groups have few pathways in common. One important lncRNA MSTRG.11108.1 and its target genes (ICAM1, CCL3, CCL4, etc.) were involved in immune-related pathways. Finally, through integrated analysis of lncRNAs with GWAS data and animal QTL database, we found that differential lncRNA and its target genes could be significantly enriched in SNPs and QTLs related to somatic cell count (SCC) and mastitis, such as MSTRG.11108.1 and its target gene ICAM1, CXCL3, GRO1.
CONCLUSIONS: For subclinical mastitic cows, folic acid supplementation can significantly affect the expression of immune-related pathway genes such as ICAM1 by regulating lncRNAs MSTRG.11108.1, thereby affecting related immune phenotypes. Our findings laid a ground foundation for theoretical and practical application for feeding folic acid supplementation in subclinical mastitic cows.

This study aimed to (1) estimate the prevalence of cow-level high somatic cell count (SCC) in Chinese dairy herds and (2) identify potential factors associated with cow- and herd-level SCC variables. The monthly data on dairy herd improvement were collected from a total of 131 dairy herds in 11 provinces in China in 2019. Mixed models were constructed using the cow composite milk SCC and the variance of cow SCC as dependent variables separately and parity, seasons, days in milk (DIM), herd size, and farm types (family-owned vs. company-owned) as fixed effects, accounting for the nested random herd and cow effect. We used negative binomial regression using herd-level SCC-related variables, namely, monthly proportion of high SCC, monthly proportion of new high SCC, monthly proportion of chronic high SCC, and monthly proportion of new chronic high SCC as dependent variables separately against seasons, herd size, and farm types with the random herd effect. The overall average prevalence of high SCCs for each month per farm was 0.26 (2.5-97.5% quantile: 0-0.56). Company-owned farms performed better in herd SCC management. Seasons were significantly associated with all the aforementioned variables, and summer and autumn were the seasons associated with worse outcomes in herd SCCs. This study is the first to assess high SCC in a large number of Chinese dairy herds, which is useful for farms to tailor the on-farm mastitis control programs in China.

Mastitis in dairy cattle is a highly prevalent infectious disease, caused by various pathogens, mainly Staphylococcu aureus and Escherichia Coli, considerable economic loss worldwide.
The aim of this study was to evaluate the in vitro activity of Herbal plants used against S. aureus and E. coli bacteria which are the causative agents of mastitis.
Therefore, in this study we investigate the antimicrobial effect of plant to evaluate the in vitro antibacterial activity of squaw mint (Mentha pulegium L., Lamiaceae family), catnip (Nepeta cataria L., Lamiaceae), lemon balm (Melissa officinalis L., Lamiaceae), for mastitis treatment. Solutions prepared in fixed oils, against S. aureus and E. coli bacteria which are the main agents of mastitis. Isolation and antibiotic susceptibility analyses of milk samples taken from 100 subclinical mastitis dairy cows were performed. The antibacterial properties of the solutions were analysed by a disk diffusion method.
In the bacterial isolation, S. aureus was determined 97.7% and E. coli 53.5% positive of cows with mastitis. Antibacterial susceptibility test of the Lemon balm extract and essential oil showed maximum zone of inhibition against S. aureus 30 µl (23 mm), followed by 20 µl (19 mm), E. coli (19 mm) and 10 µl (5-7 mm), of the same extract against the Gram-positive bacteria. The ethanol extracts show the similar activity against the Gram-negative bacteria at 30, 20, and 10 µl (18-20 mm). Followed by S. aureus, when the zone areas for the susceptible solutions (Lemon balm, and essential oil) and the control group were compared, determined that there was little difference between for S. aureus and E. coli.
This study hence indicated that in vitro cultured plantlets of lemon balm and peppermint oil can be used as the alternative method for production of mastitis and cheap source its precursor with antimicrobial activities.

Somatic cell count (SCC) is an important indicator of the health state of bovine udders. However, the exact cut-off value used for differentiating the cows with healthy quarters from the cows with subclinical mastitis remains controversial. Here, we collected composite milk (milk from four udder quarters) and peripheral blood samples from individual cows in two different dairy farms and used 16S rRNA gene sequencing combined with RNA-seq to explore the differences in the milk microbial composition and transcriptome of cows with three different SCC levels (LSCC: <100,000 cells/mL, MSCC: 100,000−200,000 cells/mL, HSCC: >200,000 cells/mL). Results showed that the milk microbial profiles and gene expression profiles of samples derived from cows in the MSCC group were indeed relatively easily discriminated from those from cows in the LSCC group. Discriminative analysis also uncovered some differentially abundant microbiota at the genus level, such as Bifidobacterium and Lachnospiraceae_AC2044_group, which were more abundant in milk samples from cows with SCC below 100,000 cells/mL. As for the transcriptome profiling, 79 differentially expressed genes (DEGs) were found to have the same direction of regulation in two sites, and functional analyses also showed that biological processes involved in inflammatory responses were more active in MSCC and HSCC cows. Overall, these results showed a similarity between the milk microbiota and gene expression profiles of MSCC and HSCC cows, which presented further evidence that 100,000 cells/ml is a more optimal cut-off value than 200,000 cells/mL for intramammary infection detection at the cow level.

Bovine mastitis, especially subclinical mastitis (SCM), is one of the most prevalent and economically costly diseases in the dairy industry worldwide. Understanding the prevalence and spatial distribution of bovine SCM and its associated risk factors will facilitate the prevention and control of the disease. We reviewed published studies pertaining to epidemiological surveys of SCM among dairy cows during the past decade (2012-2021) in China from inception to March 20, 2020, with PubMed, Clinical Trial, VIP, CNKI and databases being used to identify English and Chinese articles. Therefore, we retrieved 41 studies related to epidemiological surveys of SCM among dairy cows, using our eligibility criteria. We demonstrated that the prevalence of SCM in dairy cows was 37.7% during the selected periods, indicating a slight increase in the incidence of SCM in a comparison between 2012 and 2016 and 2017-2021. The estimated prevalence of SCM was 36.4%-50.2% in the seven regions, which was no statistically significant difference. The highest prevalence of SCM was 72% in Inner Mongolia Autonomous Region, and the lowest prevalence was 19% in Hubei Province. The variation in diagnostic methods was not significant in the studies. Dairy cows\' parity was a risk factor associated with the prevalence rate of SCM. Cow\'s age might affect the prevalence of SCM during the selected periods. This study may facilitate the control with specific strategies to reduce costs and antibiotics overuse, enhance food safety and public health.

The occurrence and development of mastitis is linked to dysbiostic gastrointestinal microbiota. Inulin is a dietary prebiotic that improves the profile of intestinal flora. Our previous study showed that inulin supplementation could improve the ruminal microbes of subclinical mastitis (SCM) cows. The current study attempted to further investigate the response of hindgut (fecal) microbiome and metabolites, serum metabolism, and protein expression to inulin in the in SCM cows. Different levels of inulin (0, 100, 200, 300, and 400 g/day per cow) were supplemented in SCM cows. Compared with control group, Bacteroides and Bifidobacteria were increased, and Paeniclostridium, Ruminococcaceae, Coprococcus, and Clostridia were decreased in the feces of inulin groups, and accompanied with elevated propionate and butyrate concentrations, while secondary bile acid (SBA) metabolites were increased and proinflammatory lipid oxidation products were dropped in both feces and serum. In serum, inulin intake suppressed the levels of triglyceride (TG) and low-density lipoprotein (LDL). Serum proteome analysis found that CD44 antigen, phosphatidylinositol-glycan-specific phospholipase D, apolipoprotein A-II, and superoxide dismutase [Cu-Zn] were upregulated, while cathelicidin-1, haptoglobin, serpin A3, inter-alpha-trypsin inhibitor heavy chain H4 were downregulated in inulin groups. These findings suggested further evidence for inulin supplementation in amelioration of inflammatory symptoms in SCM cows, which might provide alternative treatment for mastitis.

A novel ELISA test has been developed to detect antigen-specific IgG in early and late lactation cows in New Zealand.
This study was to evaluate the discriminatory ability of the ELISA based on the detection of S. aureus-specific IgG as a screening test.
The ELISA was used for the composite milk samples taken during routine herd testing in 2018-2019 milking season in New Zealand. In the absence of a gold standard test, the diagnostic specificity and sensitivity was estimated using a Gaussian mixture model.
The ELISA test had a high accuracy (AUC = 0.98) to detect antigen-specific IgG in early and late lactation cows with high somatic cell count due to either subsequent to or contemporaneous with the S. aureus invasion. Using an S/P ratio = 0.3 as the cut-off value, the ELISA test has sensitivity of 0.9 and specificity of 0.95, while the sensitivity increased to 0.94 at a cost of a decreased specificity of 0.9 at a lower cut-off value 0.26.
The integration of the ELISA test as a screening tool into specific control programs may be useful to reduce the spread of S. aureus infections, to aid with treatment decisions, and to establish a correct milking order.