An Explainable Deep Learning Classifier of Bovine Mastitis Based on Whole-Genome Sequence Data-Circumventing the p >> n Problem.

The serious drawback underlying the biological annotation of whole-genome sequence data is the p >> n problem, which means that the number of polymorphic variants (p) is much larger than the number of available phenotypic records (n). We propose a way to circumvent the problem by combining a LASSO logistic regression with deep learning to classify cows as susceptible or resistant to mastitis, based on single nucleotide polymorphism (SNP) genotypes. Among several architectures, the one with 204,642 SNPs was selected as the best. This architecture was composed of two layers with, respectively, 7 and 46 units per layer implementing respective drop-out rates of 0.210 and 0.358. The classification of the test data resulted in AUC = 0.750, accuracy = 0.650, sensitivity = 0.600, and specificity = 0.700. Significant SNPs were selected based on the SHapley Additive exPlanation (SHAP). As a final result, one GO term related to the biological process and thirteen GO terms related to molecular function were significantly enriched in the gene set that corresponded to the significant SNPs. Our findings revealed that the optimal approach can correctly predict susceptibility or resistance status for approximately 65% of cows. Genes marked by the most significant SNPs are related to the immune response and protein synthesis.

Cows with diverging haplotypes show differences in differential milk cell count, milk parameters and vaginal temperature after S. aureus challenge but not after E. coli challenge.

BACKGROUND: In dairy cattle, mastitis causes high financial losses and impairs animal well-being. Genetic selection is used to breed cows with reduced mastitis susceptibility. Techniques such as milk cell flow cytometry may improve early mastitis diagnosis. In a highly standardized in vivo infection model, 36 half-sib cows were selected for divergent paternal Bos taurus chromosome 18 haplotypes (Q vs. q) and challenged with Escherichia coli for 24 h or Staphylococcus aureus for 96 h, after which the samples were analyzed at 12 h intervals. Vaginal temperature (VT) was recorded every three minutes. The objective of this study was to compare the differential milk cell count (DMCC), milk parameters (fat %, protein %, lactose %, pH) and VT between favorable (Q) and unfavorable (q) haplotype cows using Bayesian models to evaluate their potential as improved early indicators of differential susceptibility to mastitis. RESULTS: After S. aureus challenge, compared to the Q half-sibship cows, the milk of the q cows exhibited higher PMN levels according to the DMCC (24 h, p < 0.001), a higher SCC (24 h, p < 0.01 and 36 h, p < 0.05), large cells (24 h, p < 0.05) and more dead (36 h, p < 0.001) and live cells (24 h, p < 0.01). The protein % was greater in Q milk than in q milk at 0 h (p = 0.025). In the S. aureus group, Q cows had a greater protein % (60 h, p = 0.048) and fat % (84 h, p = 0.022) than q cows. Initially, the greater VT of S. aureus-challenged q cows (0 and 12-24 h, p < 0.05) reversed to a lower VT in q cows than in Q cows (48-60 h, p < 0.05). Additionally, the following findings emphasized the validity of the model: in the S. aureus group all DMCC subpopulations (24 h-96 h, p < 0.001) and in the E. coli group nearly all DMCC subpopulations (12 h-24 h, p < 0.001) were higher in challenged quarters than in unchallenged quarters. The lactose % was lower in the milk samples of E. coli-challenged quarters than in those of S. aureus-challenged quarters (24 h, p < 0.001). Between 12 and 18 h, the VT was greater in cows challenged with E. coli than in those challenged with S. aureus (3-h interval approach, p < 0.001). CONCLUSION: This in vivo infection model confirmed specific differences between Q and q cows with respect to the DMCC, milk component analysis results and VT results after S. aureus inoculation but not after E. coli challenge. However, compared with conventional milk cell analysis monitoring, e.g., the global SCC, the DMCC analysis did not provide refined phenotyping of the pathogen response.

The use of stem cells in the treatment of mastitis in dairy cows.

Mastitis is a multifactorial inflammatory disease. The increase in antibiotic resistance of bacteria that cause mastitis means that cattle breeders would prefer to reduce the use of antibiotics. Recently, therapies using mesenchymal stem cells (MSCs) from various sources have gained significant interest in the development of regenerative medicine in humans and animals, due to their extraordinary range of properties and functions. The aim of this study was to analyze the effectiveness of an allogeneic stem cells derived from bone marrow (BMSC) and adipose tissue (ADSC) in treating mastitis in dairy cattle. The research material consisted of milk and blood samples collected from 39 Polish Holstein-Friesian cows, 36 of which were classified as having mastitis, based on cytological evaluation of their milk. The experimental group was divided into subgroups according to the method of MSC administration: intravenous, intramammary, and intravenous + intramammary, and according to the allogeneic stem cells administered: BMSC and ADSC. The research material was collected at several time intervals: before the administration of stem cells, after 24 and 72 h, and after 7 days. Blood samples were collected to assess hematological parameters and the level of pro-inflammatory cytokines, while the milk samples were used for microbiological assessment and to determine the somatic cells count (SCC). The administration of allogeneic MSCs resulted in a reduction in the total number of bacterial cells, Staphylococcus aureus, bacteria from the Enterobacteriaceae group, and a systematic decrease in SCC in milk. The therapeutic effect was achieved via intravenous + intramammary or intramammary administration.

Genetic diversity and phylogenetic relationships of Clostridium perfringens strains isolated from mastitis and enteritis in Egyptian dairy farms.

BACKGROUND: Clostridium perfringens, a common environmental bacterium, is responsible for a variety of serious illnesses including food poisoning, digestive disorders, and soft tissue infections. Mastitis in lactating cattle and sudden death losses in baby calves are major problems for producers raising calves on dairy farms. The pathogenicity of this bacterium is largely mediated by its production of various toxins. RESULTS: The study revealed that Among the examined lactating animals with a history of mastitis, diarrheal baby calves, and acute sudden death cases in calves, C. perfringens was isolated in 23.5% (93/395) of the total tested samples. Eighteen isolates were obtained from mastitic milk, 59 from rectal swabs, and 16 from the intestinal contents of dead calves. Most of the recovered C. perfringens isolates (95.6%) were identified as type A by molecular toxinotyping, except for four isolates from sudden death cases (type C). Notably, C. perfringens was recovered in 100% of sudden death cases compared with 32.9% of rectal swabs and 9% of milk samples. This study analyzed the phylogeny of C. perfringens using the plc region and identified the plc region in five Egyptian bovine isolates (milk and fecal origins). Importantly, this finding expands the known data on C. perfringens phospholipase C beyond reference strains in GenBank from various animal and environmental sources. CONCLUSION: Phylogenetic analyses of nucleotide sequence data differentiated between strains of different origins. The plc sequences of Egyptian C. perfringens strains acquired in the present study differed from those reported globally and constituted a distinct genetic ancestor.

Lycopene promoted M2 macrophage polarization via inhibition of NOTCH1-PI3K-mTOR-NF-κB-JMJD3-IRF4 pathway in response to Escherichia coli infection in J744A.1 cells.

Escherichia coli (E. coli) can induce severe clinical bovine mastitis, which is to blame for large losses experienced by dairy farms. Macrophage polarization into various states is in response to pathogen infections. Lycopene, a naturally occurring hydrocarbon carotenoid, relieved inflammation by controlling M1/M2 status of macrophages. Thus, we wanted to explore the effect of lycopene on polarization states of macrophages in E. coli-induced mastitis. Macrophages were cultivated with lycopene for 24, before E. coli inoculation for 6 h. Lycopene (0.5 µmol/L) significantly enhanced cell viabilities and significantly reduced lactic dehydrogenase (LDH) levels in macrophages, whereas 2 and 3 µmol/L lycopene significantly enhanced LDH activities. Lycopene treatment significantly reduced the increase in LDH release, iNOS, CD86, TNF-α, IL-1ß and phosphatase and tensin homolog (PTEN) expressions in E. coli group. 0.5 µmol/L lycopene significantly increased E. coli-induced downregulation of CD206, arginase I (ARG1), indoleamine 2,3-dioxygenase (IDO), chitinase 3-like 3 (YM1), PI3K, AKT, p-AKT, mammalian target of rapamycin (mTOR), p-mTOR, jumonji domain-containing protein-3 (JMJD3) and interferon regulatory factor 4 (IRF4) levels. Moreover, Ginkgolic acid C17:1 (a specific PTEN inhibitor), 740YPDGFR (a specific PI3K activator), SC79 (a specific AKT activator) or CHPG sodium salt (a specific NF-κB activator) significantly decreased CD206, AGR1, IDO and YM1 expressions in lycopene and E. coli-treated macrophages. Therefore, lycopene increased M2 macrophages via inhibiting NOTCH1-PI3K-mTOR-NF-κB-JMJD3-IRF4 pathway in response to E. coli infection in macrophages. These results contribute to revealing the pathogenesis of E. coli-caused bovine mastitis, providing the new angle of the prevention and management of mastitis.

[Investigations of a controlled, decision tree based procedure for Selective Dry Cow Treatment in Bavarian dairy farms]. / Untersuchungen zu einem kontrollierten, entscheidungsbaumbasierten Verfahren des Selektiven Trockenstellens in Bayerischen Milchviehbetrieben.

OBJECTIVE: Four parameters of a decision tree for Selective Dry Cow Treatment (SDCT), examined in a previous study, were analyzed regarding their efficacy in detecting cows for dry cow treatment (DCT, use of intramammary antimicrobials). This study set out to review wether all parameters (somatic cell count [SCC≥ 200 000 SC/ml 3 months' milk yield recordings prior dry off (DO)], clinical mastitis history during lactation [≥1 CM], culturing [14d prior DO, detection of major pathogens] and California-Mastitis-Test [CMT, > rate 1/+ at DO]) are necessary for accurate decision making, whether there are possible alternatives to replace culturing, and whether a simplified model could replace the decision tree. MATERIAL AND METHODS: Records of 18 Bavarian dairy farms from June 2015 to August 2017 were processed. Data analysis was carried out by means of descriptive statistics, as well as employing a binary cost sensitive classification tree and logit-models. For statistical analyses the outcomes of the full 4-parameter decision tree were taken as ground truth. RESULTS: 848 drying off procedures in 739 dairy cows (CDO) were included. SCC and CMT selected 88.1%, in combination with CM 95.6% of the cows that received DCT (n=494). Without culturing, 22 (4.4%) with major pathogens (8x Staphylococcus [S.] aureus) infected CDO would have been misclassified as not needing DCT. The average of geometric mean SCC (within 100 d prior DO) for CDO with negative results in culturing was<100 000 SC/ml milk, 100 000-150 000 SC/ml for CDO infected with minor pathogens, and ≥ 150 000 SC/ml for CDO infected with major pathogens (excluding S.aureus). Using SCC during lactation (at least 1x > 200 000 SC/ml) and positive CMT to select CDO for DCT, contrary to the decision tree, 37 CDO (4.4%) would have been treated "incorrectly without" and 43 CDO (5.1%) "unnecessarily with" DCT. Modifications were identified, such as SCC<131 000 SC/ml within 100 d prior to DO for detecting CDO with no growth or minor pathogens in culturing. The best model for grading CDO for or against DCT (CDO without CM and SCC<200 000 SC/ml [last 3 months prior DO]) had metrics of AUC=0.74, Accuracy=0.778, balanced Accuracy=0.63, Sensitivity=0.92 and Specificity=0.33. CONCLUSIONS: Combining the decision tree's parameters SCC, CMT and CM renders suitable selection criteria under the conditions of this study. When omitting culturing, lower thresholds for SCC should be considered for each farm individually to select CDO for DCT. Nonetheless, the most accurate model could not replace the full decision tree.

Development of a prototypic, field-usable diagnostic tool for the detection of gram-positive cocci-induced mastitis in cattle.

BACKGROUND: Bovine mastitis is one of the most widespread diseases affecting cattle, leading to significant losses for the dairy industry. Currently, the so-called gold standard in mastitis diagnosis involves determining the somatic cell count (SCC). Apart from a number of advantages, this method has one serious flaw: It does not identify the etiological factor causing a particular infection, making it impossible to introduce targeted antimicrobial therapy. This can contribute to multidrug-resistance in bacterial species. The diagnostic market lacks a test that has the advantages of SCC and also recognizes the species of pathogen causing the inflammation. Therefore, the aim of our study was to develop a lateral flow immunoassay (LFIA) based on elongation factor Tu for identifying most prevalent Gram-positive cocci responsible for causing mastitis including Streptococcus uberis, Streptococcus agalactiae and Staphylococcus aureus. RESULTS: As a result, we showed that the assay for S. uberis detection demonstrated a specificity of 89.02%, a sensitivity of 43.59%, and an accuracy of 80.3%. In turn, the second variant - assay for Gram-positive cocci reached a specificity of 95.59%, a sensitivity of 43.28%, and an accuracy of 78.33%. CONCLUSIONS: Our study shows that EF-Tu is a promising target for LFIA and we have delivered evidence that further evaluation could improve test parameters and fill the gap in the mastitis diagnostics market.

Molecular and phenotypic identification of bacterial species isolated from cows with mastitis from three regions of Poland.

BACKGROUND: Bovine mastitis is a widespread disease affecting dairy cattle worldwide and it generates substantial losses for dairy farmers. Mastitis may be caused by bacteria, fungi or algae. The most common species isolated from infected milk are, among others, Streptococcus spp., Escherichia coli, Staphylococcus aureus and non-aureus staphylococci and mammaliicocci. The aim of this paper is to determine the frequency of occurrence of bacterial species in milk samples from cows with mastitis from three regions of Poland: the north-east, the south-west and the south. To this end 203 milk samples taken from cows with a clinical form (CM) of mastitis (n = 100) and healthy animals (n = 103) were examined, which included culture on an appropriate medium followed by molecular detection of E. coli, S. aureus, Streptococcus agalactiae and Streptococcus uberis, as one of the most common species isolated from mastitis milk. RESULTS: The results obtained indicated that S. uberis was the most commonly cultivated CM species (38%, n = 38), followed by S. aureus (22%, n = 22), E. coli (21%, n = 21) and S. agalactiae (18%, n = 18). Similar frequencies in molecular methods were obtained for S. uberis (35.1%) and S. aureus (28.0%). The variation of sensitivity of both methods may be responsible for the differences in the E. coli (41.0%, p = 0.002) and S. agalactiae (5.0%, p = 0.004) detection rates. Significant differences in composition of species between three regions of Poland were noted for E. coli incidence (p < 0.001), in both the culture and molecular methods, but data obtained by the PCR method indicated that this species was the least common in north-eastern Poland, while the culture method showed that in north-eastern Poland E. coli was the most common species. Significant differences for the molecular method were also observed for S. uberis (p < 0.001) and S. aureus (p < 0.001). Both species were most common in southern and south-western Poland. CONCLUSIONS: The results obtained confirm the need to introduce rapid molecular tests for veterinary diagnostics, as well as providing important epidemiological data, to the best of our knowledge data on Polish cows in selected areas of Poland is lacking.

Screening of essential oils against oxacillin - resistant Staphylococcus aureus strains isolated from bovine mastitis / Screening de aceites esenciales frente a Staphylococcus aureus oxacilino - resistentes extraidos de aislados de mastitis bovina

Bovine mastitis is a disease wi th far - reaching consequences for the dairy industry. Staphylococcus aureus is a pathogen that is especially resistant to antibiotics. The objective of this study was to evaluate the antimicrobial activity of the essential oils Lippia citriodora (Lam.), Thy mus vulgaris (L), and a mixture of the essential oils Lippia citriodora and Thymus vulgaris (50/50 v/v), against isolates of oxacillin - resistant Staphylococcus aureus (n=15) of positive cases of bovine mastitis. For the statistical analysis, the IBM SPSS s tatistical package was used. The mixture of essential oils ( Lippia citriodora and Thymus vulgaris (50/50 v/v)) obtained the most significant antimicrobial activity in relation to pure essential oils. It is therefore concluded that the mixture of these oils boosts their antimicrobial activity ( p <0.05). The minimum inhibitory and bactericidal concentration of this mixture for the total isolations was 12 µL/L and 25 µL/mL, respectively.

La mastitis bovina es una enfermedad de gran impacto para la industria lechera. El Staphylococcus aureus es uno de los principales patógenos, especialmente aquellos resistentes a los antibióticos. El objetivo de este estudio fue evaluar la actividad antimicrobiana de los aceites esenciales de Lippia citriodora (Lam.), Thymus vulgaris (L), y una mezcla de aceites esenciales de Lippia citriodora y Thymus vulgaris (50/50 v/v), frente a aislamientos clínicos de Staph ylococcus aureus oxacilino - resistentes (n=15) de mastitis bovina. Se utilizó p rograma estadístico IBM SPSS y se concluyó la diferencia significativa a un p <0.05. La mezcla de aceites esenciales ( Lippia citriodora y Thymus vulgaris (50/50 v/v)), obtuvo la m ayor actividad antimicrobiana en relación a los aceites esenciales puros, se concluye que la mezcla de estos aceites potencia su actividad antimicrobiana ( p <0.019). La concentración mínima inhibitoria y bactericida de esta mezcla fue del 12 µL/mL y 25 µL/m L, respectivamente, y puede ser una alternativa terapéutica.

Biomarker and proteome analysis of milk from dairy cows with clinical mastitis: Determining the effect of different bacterial pathogens on the response to infection.

Antimicrobial usage (AMU) could be reduced by differentiating the causative bacteria in cases of clinical mastitis (CM) as either Gram-positive or Gram-negative bacteria or identifying whether the case is culture-negative (no growth, NG) mastitis. Immunoassays for biomarker analysis and a Tandem Mass Tag (TMT) proteomic investigation were employed to identify differences between samples of milk from cows with CM caused by different bacteria. A total of 94 milk samples were collected from cows diagnosed with CM across seven farms in Scotland, categorized by severity as mild (score 1), moderate (score 2), or severe (score 3). Bovine haptoglobin (Hp), milk amyloid A (MAA), C-reactive protein (CRP), lactoferrin (LF), α-lactalbumin (LA) and cathelicidin (CATHL) were significantly higher in milk from cows with CM, regardless of culture results, than in milk from healthy cows (all P-values <0.001). Milk cathelicidin (CATHL) was evaluated using a novel ELISA technique that utilises an antibody to a peptide sequence of SSEANLYRLLELD (aa49-61) common to CATHL 1-7 isoforms. A classification tree was fitted on the six biomarkers to predict Gram-positive bacteria within mastitis severity scores 1 or 2, revealing that compared to the rest of the samples, Gram-positive samples were associated with CRP < 9.5 µg/ml and LF ≥ 325 µg/ml and MAA < 16 µg/ml. Sensitivity of the tree model was 64%, the specificity was 91%, and the overall misclassification rate was 18%. The area under the ROC curve for this tree model was 0.836 (95% bootstrap confidence interval: 0.742; 0.917). TMT proteomic analysis revealed little difference between the groups in protein abundance when the three groups (Gram-positive, Gram-negative and no growth) were compared, however when each group was compared against the entirety of the remaining samples, 28 differentially abundant protein were identified including ß-lactoglobulin and ribonuclease. Whilst further research is required to draw together and refine a suitable biomarker panel and diagnostic algorithm for differentiating Gram- positive/negative and NG CM, these results have highlighted a potential panel and diagnostic decision tree. Host-derived milk biomarkers offer significant potential to refine and reduce AMU and circumvent the many challenges associated with microbiological culture, both within the lab and on the farm, while providing the added benefit of reducing turnaround time from 14 to 16 h of microbiological culture to just 15 min with a lateral flow device (LFD).

Antimicrobial resistance patterns of Streptococcus uberis isolates from bovine milk in Chiba prefecture, Japan: association between multidrug resistance and clonal complex 996.

Streptococcus uberis is one of major pathogens causing bovine mastitis. However, there is poor information on antimicrobial resistance (AMR) among the Japanese isolates. To provide treatment information for the mastitis caused by S. uberis in Japan, we aimed to clarify AMR patterns of the isolates from bovine milk mainly in Chiba. AMR phenotyping/genotyping [blaZ-erm(A)-erm(B)-mef(A)-linB-lnuD-tet(M)-tet(O)-tet(K)-tet(L)-tet(S)] and multilocus sequence typing were performed to analyze relationships between AMR patterns and clonal complexes (CCs). Resistance to tetracycline-, macrolide-, and lincosamide-classes was mainly associated with possession of tet(O), tet(S), erm(B), linB, and lnuD genes. CC996 was significantly associated with multidrug resistance (P<0.0001). These findings will aid Chiba farm animal clinics in treating bovine mastitis.

Milk miRNA expression in buffaloes as a potential biomarker for mastitis.

BACKGROUND: Buffaloes have the highest potential for production due to a promising gene pool that is being enhanced and upgraded. Mastitis is a significant health impediment that greatly diminishes milk yield and quality, affecting rural farmers' livelihoods. The traditional gold standard used for diagnosing mastitis or subclinical mastitis is CMT, but it has the drawback of false positive or negative results. Subclinical mastitis, if not treated promptly, can lead to mammary tumors. To address the gap in early diagnosis of subclinical mastitis in CMT-negative milk of buffaloes, we performed a retrospective analysis and evaluated the milk miRNA expression profiles as potential biomarkers. RESULTS: Thirty buffalo milk samples based on clinical signs and CMT were divided into normal, subclinical, and clinical mastitis. SCC evaluation showed significant differences between the groups. The data analysis demonstrated that the elevation of miR-146a and miR-383 differed substantially between normal, subclinical, and clinical mastitis milk of buffaloes with 100% sensitivity and specificity. The relationship of SCC with miR-146a and miR-383 in normal/healthy and subclinical mastitis was positively correlated. CONCLUSION: The overexpression of miR-146a and miR-383 is associated with inflammation. It can be a valuable prognostic and most sensitive biomarker for early mastitis detection in buffaloes with SCC below 2 lakhs and CMT-ve, enhancing the accuracy of subclinical mastitis diagnosis.

Interaction of blood calcium with milk yield, energy metabolites, subclinical mastitis, and reproductive performance in crossbred dairy cows.

Limited literature is available on the consequences of postpartum low blood calcium (Ca) concentration in crossbred cows. The research aimed to investigate the correlation between postpartum serum Ca levels and various parameters, including milk yield, serum energy metabolites, milk somatic cell count, and reproductive factors in crossbred cows. Following parturition, a total of 45 potential high-yielding F2 (HF × Sahiwal; Genotype: 75:25) dairy cows were enrolled . These cows were categorized based on plasma calcium concentrations into three groups: a low calcium group (Ca-L) with a calcium concentration of <5 mg/dL, a medium calcium group (Ca-M) with a calcium concentration ranging from 5 to 8.5 mg/dL, and a high calcium group (Ca-H) with a calcium concentration exceeding 8.5 mg/dL. The study parameters were measured over an 8-week period. The results indicated that overall milk yield and blood glucose were significantly higher in the Ca-H group compared to Ca-M and Ca-L (p < .01). Blood cholesterol was significantly higher in Ca-M (p < .01), while blood triglyceride was significantly lower in both Ca-M and Ca-H. Overall, blood cortisol did not show a significant change between these groups (p < .01); however, progesterone levels were higher (p < .01) in Ca-M and Ca-H cows. Furthermore, somatic cell count (SCC) significantly (p < .01) decreased in cows with Ca-H compared to Ca-L. Additionally, postpartum oestrous interval and interestrus interval decreased significantly (p < .01) in Ca-M and Ca-H compared to Ca-L. These findings suggest that cows with blood calcium levels exceeding 8.5 mg/dL exhibited significantly higher milk yield, blood metabolite levels, a lower likelihood of subclinical mastitis, and earlier reproductive activity after calving.

Transcriptomic Changes and Regulatory Networks Associated with Resistance to Mastitis in Xinjiang Brown Cattle.

Xinjiang brown cattle are highly resistant to disease and tolerant of roughage feeding. The identification of genes regulating mastitis resistance in Xinjiang brown cattle is a novel means of genetic improvement. In this study, the blood levels of IL-1ß, IL-6, IL-10, TNF-α, and TGF-ß in Xinjiang brown cattle with high and low somatic cell counts (SCCs) were investigated, showing that cytokine levels were higher in cattle with high SCCs. The peripheral blood transcriptomic profiles of healthy and mastitis-affected cattle were constructed by RNA-seq. Differential expression analysis identified 1632 differentially expressed mRNAs (DE-mRNAs), 1757 differentially expressed lncRNAs (DE-lncRNAs), and 23 differentially expressed circRNAs (DE-circRNAs), which were found to be enriched in key pathways such as PI3K/Akt, focal adhesion, and ECM-receptor interactions. Finally, ceRNA interaction networks were constructed using the differentially expressed genes and ceRNAs. It was found that keynote genes or mRNAs were also enriched in pathways such as PI3K-Akt, cholinergic synapses, cell adhesion molecules, ion binding, cytokine receptor activity, and peptide receptor activity, suggesting that the key genes and ncRNAs in the network may play an important role in the regulation of bovine mastitis.

Bacillus velezensis iturins inhibit the hemolytic activity of Staphylococcus aureus.

Bovine mastitis caused by S. aureus has a major economic impact on the dairy sector. With the crucial need for new therapies, anti-virulence strategies have gained attention as alternatives to antibiotics. Here we aimed to identify novel compounds that inhibit the production/activity of hemolysins, a virulence factor of S. aureus associated with mastitis severity. We screened Bacillus strains obtained from diverse sources for compounds showing anti-hemolytic activity. Our results demonstrate that lipopeptides produced by Bacillus spp. completely prevented the hemolytic activity of S. aureus at certain concentrations. Following purification, both iturins, fengycins, and surfactins were able to reduce hemolysis caused by S. aureus, with iturins showing the highest anti-hemolytic activity (up to 76% reduction). The lipopeptides showed an effect at the post-translational level. Molecular docking simulations demonstrated that these compounds can bind to hemolysin, possibly interfering with enzyme action. Lastly, molecular dynamics analysis indicated general stability of important residues for hemolysin activity as well as the presence of hydrogen bonds between iturins and these residues, with longevous interactions. Our data reveals, for the first time, an anti-hemolytic activity of lipopeptides and highlights the potential application of iturins as an anti-virulence therapy to control bovine mastitis caused by S. aureus.

Pathogen group-specific risk factors for intramammary infection in water buffalo.

A cross-sectional study was conducted to estimate the prevalence of intramammary infection (IMI) associated bacteria and to identify risk factors for pathogen group-specific IMI in water buffalo in Bangladesh. A California Mastitis Test (CMT) and bacteriological cultures were performed on 1,374 quarter milk samples collected from 763 water buffalo from 244 buffalo farms in nine districts in Bangladesh. Quarter, buffalo, and farm-related data were obtained through questionnaires and visual observations. A total of 618 quarter samples were found to be culture positive. Non-aureus staphylococci were the predominant IMI-associated bacterial species, and Staphylococcus (S.) chromogenes, S. hyicus, and S. epidermidis were the most common bacteria found. The proportion of non-aureus staphylococci or Mammaliicoccus sciuri (NASM), S. aureus, and other bacterial species identified in the buffalo quarter samples varied between buffalo farms. Therefore, different management practices, buffalo breeding factors, and nutrition were considered and further analyzed when estimating the IMI odds ratio (OR). The odds of IMI by any pathogen (OR: 1.8) or by NASM (OR: 2.2) was high in buffalo herds with poor milking hygiene. Poor cleanliness of the hind quarters had a high odds of IMI caused by any pathogen (OR: 2.0) or NASM (OR: 1.9). Twice daily milking (OR: 3.1) and farms with buffalo purchased from another herd (OR: 2.0) were associated with IMI by any pathogen. Asymmetrical udders were associated with IMI-caused by any bacteria (OR: 1.7). A poor body condition score showed higher odds of IMI by any pathogen (OR: 1.4) or by NASM (OR: 1.7). This study shows that the prevalence of IMI in water buffalo was high and varied between farms. In accordance with the literature, our data highlight that IMI can be partly controlled through better farm management, primarily by improving hygiene, milking management, breeding, and nutrition.

Cow-level risk factors associated with the increase in somatic cell count and the occurrence of subclinical mastitis in Brazilian Holstein and Jersey dairy cows.

Mastitis, a multifactorial disease influenced by both cow and herd-level factors, results in significant losses throughout the dairy chain. We aimed to evaluate the relationship between milking frequency (MF), parity order (PO), days in milk (DIM), and milk yield (MY) on somatic cell count (SCC) and the odds of a cow having subclinical mastitis (SCM) in Brazilian Holstein and Jersey dairy cows. Our dataset consisted of 747,520 test-day records from 52,954 cows, including 49,089 Holstein cows and 3865 Jersey cows and 498 herds. The SCC was evaluated using a generalized linear mixed model, whereas SCM occurrence was evaluated using a logistic regression model. A case of SCM was defined when a cow had >200×103 cells/mL. Our results indicated that the SCC increases with higher PO and DIM and decreases in cows milked three times a day and those with higher MY in both breeds (>40 and >25 L/d for Holstein and Jersey, respectively). Increasing MF from two to three times a day reduced the chances of a Holstein and Jersey cow having SCM by 10 and 20 %, respectively. For Holstein and Jersey cows, those with ≥quadriparous had 3.9 times and 2.2 times higher chances, respectively, of having SCM compared to primiparous cows. Cows with >305 DIM had 2.0 times greater chances of having SCM for both, Holstein and Jersey cows, compared to cows with ≤105 DIM. Holstein cows yielding ≥40 L/d had a 75 % lower chance of having SCM compared to those yielding <20 L/d, while Jersey cows with ≥25 L/d had a 60 % lower chance compared to those yielding <15 L/d. In conclusion, higher PO and DIM pose risks, whereas a MF of three times a day and higher MY are protective factors against increases in SCC and SCM occurrence in Brazilian Holstein and Jersey cows.

Antimicrobial resistance of Enterobacteriaceae and Staphylococcus spp. isolated from raw cow's milk from healthy, clinical and subclinical mastitis udders.

Mastitis is the most common disease of dairy cattle and can be manifested in clinical and subclinical forms. The overuse of antimicrobials in the treatment and prevention of mastitis favours antimicrobial resistance and milk can be a potential route of dissemination. This study aimed to evaluate the biological quality of bulk tank milk (BTM) and the microbiological quality and signs of mastitis of freshly milked raw milk. In addition, to evaluate antimicrobial resistance in Enterobacteriaceae and Staphylococcus spp. isolated from freshly milked raw milk. None of the farms were within the official Brazilian biological quality limits for BTM. Freshly milked raw milk with signs of clinical (CMM), subclinical (SCMM) and no signs (MFM) of mastitis were detected in 6.67%, 27.62% and 65.71% samples, respectively. Most samples of freshly milked raw milk showed acceptable microbiological quality, when evaluating the indicators total coliforms (78.10%), Escherichia coli (88.57%) and Staphylococcus aureus (100%). Klebsiella oxytoca and S. aureus were the most prevalent microorganisms in SCMM and MFM samples. Antimicrobial resistance and multidrug resistance (MDR) were observed in 65.12% and 13.95% of Enterobacteriaceae and 84.31% and 5.88% of Staphylococcus spp., respectively, isolated from both SCMM and MFM samples. Enterobacteriaceae resistant to third-generation cephalosporin (3GCR) (6.98%) and carbapenems (CRE) (6.98%) and methicillin-resistant S. aureus (MRSA) (4.88%) were observed. Antimicrobial-resistant bacteria can spread resistance genes to previously susceptible bacteria. This is a problem that affects animal, human and environmental health and should be evaluated within the one-health concept.

Identification of candidate genes associated with milk production and mastitis based on transcriptome-wide association study.

Genetic research for the assessment of mastitis and milk production traits simultaneously has a long history. The main issue that arises in this context is the known existence of a positive correlation between the risk of mastitis and lactation performance due to selection. The transcriptome-wide association study (TWAS) approach endeavors to combine the expression quantitative trait loci and genome-wide association study summary statistics to decode complex traits or diseases. Accordingly, we used the farmgtex project results as a complete bovine database for mastitis and milk production. The results of colocalization and TWAS approaches were used for the detection of functional associated candidate genes with milk production and mastitis traits on multiple tissue-based transcriptome records. Also, we used the david database for gene ontology to identify significant terms and associated genes. For the identification of interaction networks, the genemania and string databases were used. Also, the available z-scores in TWAS results were used for the calculation of the correlation between tissues. Therefore, the present results confirm that LYNX1, DGAT1, C14H8orf33, and LY6E were identified as significant genes associated with milk production in eight, six, five, and five tissues, respectively. Also, FBXL6 was detected as a significant gene associated with mastitis trait. CLN3 and ZNF34 genes emerged via both the colocalization and TWAS approaches as significant genes for milk production trait. It is expected that TWAS and colocalization can improve our perception of the potential health status control mechanism in high-yielding dairy cows.

Staphylococcus aureus promotes its intracellular survival by inhibiting Rab11-Rab11FIP4-mediated vesicle trafficking.

Mastitis in dairy cows is mainly caused by bacteria, in which Staphylococcus aureus appears frequently. Epithelial cells, as a major physical barrier of mammary gland, play an important role in preventing mastitis in dairy cows. Our previous study reported that Rab11fip4 (an effector of Rab11) was significantly changed in response to stimulation by S. aureus. So, in this study, the role of Rab11A in phagocytosis of bovine mammary epithelial cells (MAC-T) against S. aureus was evaluated. First, changes of Rab11A and Rab11fip4 were analyzed in response to S. aureus by immunofluorescence and western blotting. Subsequently, the effects of Rab11A and Rab11fip4 on proliferation of S. aureus, as well as formation and function of late endosomes (LEs) and lysosomes (LYSs) were investigated. The results showed that, after infection, Rab11A and Rab11fip4 were recruited to phagosomes containing S. aureus. Rab11A promoted bacterial clearance and rescues the destruction of LEs and LYSs by S. aureus, whereas Rab11fip4 did the opposite. These findings provide new insights into phagocytosis and control of S. aureus in host cells, thus lay the foundation to elucidate the pathogenesis of S. aureus in bovine mastitis.