Droplet digital PCR quantification of selected microRNAs in raw mastitic cow's milk from the west of Poland.

Introduction: MicroRNAs (miRNAs), a class of noncoding small RNAs, have been recognised as potential biomarkers of mammary gland conditions, including bovine mastitis diagnosis. The aim of this study was to quantify selected miRNAs in the milk of mastitic cows. Material and Methods: Milk samples (n = 90) were collected from healthy and mastitic dairy cows originating from local dairy cattle farms located in the west of Poland. MicroRNAs of the miR-21a, miR-92a, miR-146a and miR-383 species were quantified using the highly sensitive droplet digital PCR method. Direct measurement of somatic cell count (SCC) was performed using a cell counter. Cows were divided into three groups: those with an SCC below 200,000/mL were designated Low (n = 25), those with an SCC between 200,000 and 999,999 were Medium (n = 34), and those with an SCC of 1,000,000 or higher were High (n = 31). Microbiological analyses were performed using standard culture testing. Results: The level of miR-383 was very low and this miRNA was excluded from analysis. The miR-92a was used to normalise miR-21a and miR-146a expression levels. The obtained results of expression of miR-21a and miR-146a correlated with somatic cell number (R = 0.53 and 0.79, respectively). Conclusion: These results show that ddPCR is a useful method for quantifying miRNAs in raw cow milk. It seems that miR-146a is a promising marker for bovine mastitis, although further studies are needed to select a panel of miRNAs that can be used in mastitis monitoring in Poland.

In Vitro Studies of Nanoparticles as a Potentially New Antimicrobial Agent for the Prevention and Treatment of Lameness and Digital Dermatitis in Cattle.

Digital dermatitis (DD) is the second most prevalent disease in dairy cattle. It causes significant losses for dairy breeders and negatively impacts cows' welfare and milk yield. Despite this, its etiology has not been entirely identified, and available data are limited. Antibiotic therapy is a practical method for managing animal health, but overuse has caused the evolution of antibiotic-resistant bacteria, leading to a loss in antimicrobial efficacy. The antimicrobial properties of metal nanoparticles (NPs) may be a potential alternative to antibiotics. The aim of this study was to determine the biocidal properties of AgNPs, CuNPs, AuNPs, PtNPs, FeNPs, and their nanocomposites against pathogens isolated from cows suffering from hoof diseases, especially DD. The isolated pathogens included Sphingomonas paucimobilis, Ochrobactrum intermedium I, Ochrobactrum intermedium II, Ochrobactrum gallinifaecis, and Actinomyces odontolyticus. Cultures were prepared in aerobic and anaerobic environments. The viability of the pathogens was then determined after applying nanoparticles at various concentrations. The in vitro experiment showed that AgNPs and CuNPs, and their complexes, had the highest biocidal effect on pathogens. The NPs' biocidal properties and their synergistic effects were confirmed, which may forecast their use in the future treatment and the prevention of lameness in cows, especially DD.

Silver and Copper Nanoparticles as the New Biocidal Agents Used in Pre- and Post-Milking Disinfectants with the Addition of Cosmetic Substrates in Dairy Cows.

Mastitis is one of the most common issues for milk producers around the world. Antibiotic therapy is often ineffective, and therefore, scientists must find a new solution. The aim of this paper is to estimate the influence of common and well-known cosmetic substrates and mixtures of nanoparticles (NPs) and cosmetic substrates on the viability of frequently occurring mastitis pathogens, Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The obtained results suggest that only collagen + elastin and glycerine influenced and increased bacteria viability. In case of the rest of the cosmetic substrates, the viability of E. coli and S. aureus was decreased, and the results were statistically significant (p ≤ 0.01). Prepared pre-dipping and dipping mixtures decrease (p ≤ 0.01) the viability of the mentioned pathogens. The obtained results of the in vitro analysis are very promising. In the next step, prepared mixtures should be tested in different herd conditions if they can be used in mastitis prevention or decrease the number of subclinical mastitis cases. Furthermore, these mixtures could become an interesting alternative for organic milk production where conventional preparations and antibiotics are forbidden. However, further analysis, especially on the influence of prepared mixtures on other bacteria species and, algae, fungi, are necessary.

Different European Perspectives on the Treatment of Clinical Mastitis in Lactation.

As part of the European Network for Optimization of Veterinary Antimicrobial Treatment (ENOVAT), a webinar on the topic "Mastitis Treatment in Lactation" was held, in which eight mastitis experts from different European countries (Spain, The Netherlands, Estonia, Ireland, Poland, Finland, Germany, and Italy) presented their treatment approaches for clinical mastitis in lactation. The aim of this study was to compare the therapeutic approaches to identify commonalities and differences. In all eight participating countries, the decision to start treatment is usually made by the veterinarians, while the farm personnel are responsible for treatment administration. Antibiotic treatment is then typically administered intramammarily. The treatment duration often depends on the label instructions and is frequently extended if Staphylococcus aureus or Streptococcus uberis is involved. Administering supportive therapy, especially non-steroidal anti-inflammatory drugs (NSAIDs) is an established practice in all countries. Penicillin is the first-choice drug for the treatment of mastitis in an increasing number of countries. The use of critically important antimicrobials (CIAs) such as quinolones and third- and fourth-generation cephalosporins is at a low level in Finland and The Netherlands. In Estonia, Germany, Italy, and Spain, the use of CIAs is declining and is only allowed if milk samples are analyzed in advance following the legal framework. Systems for monitoring antibiotic use are being introduced in more and more countries. This exchange of different views will help the European countries to move towards a common high standard of antimicrobial stewardship in veterinary medicine.

Different methods of eubiotic feed additive provision affect the health, performance, fermentation, and metabolic status of dairy calves during the preweaning period.

BACKGROUND: This study aimed to evaluate whether different methods of providing eubiotic feed additives to neonatal calves, during the preweaning period, can improve the calves' health, performance, ruminal fermentation, and metabolic status. Forty-four (3-day-old) Holstein-Friesian dairy calves (22 female and 22 male) were divided into four treatment groups for the duration of the 8-week trial. The eubiotic feed additive consisted of a combination of probiotic Lactobacillus spp. (multiple-strains at a dose of 250 mg/calf/day) and phytobiotics containing rosmarinic acid, as the main bioactive compound (at a dose of 50 mg/calf/day). The groups were named: CON (control, without eubiotic in either the milk replacer or the starter feed), MR (eubiotic in the milk replacer), SF (eubiotic in the starter feed), MRS (eubiotic in both the milk replacer and the starter feed). The individual intake of starter feed and the fecal scores were measured daily, and body weight and biometric measurements were taken weekly until calves were 56 days of age. Blood samples were collected on day 3 and then every 14 days to determine concentrations of insulin-like-growth-factor-I, ß-hydroxybutyrate, non-esterified fatty acids, and blood urea nitrogen. Ruminal fluid was collected on days 28 and 56 for short-chain fatty acids, NH3-N, and pH measurements. RESULTS: The body weight of the calves of the MR treatment group was higher compared to all other groups on days 28 and 56. Including the eubiotic feed additive in the milk replacer increased average daily gain, starter intake, and total dry matter intake from day 29 to day 56 and the overall experimental period compared to the CON group. The calves with MR treatment had lower fecal scores from days 3 to 28, a number of parasite oocysts/cysts per gram of feces on day 28, and the occurrences of fecal consistency scores of 3 (mild diarrhea) and 4 (severe diarrhea) were 3.2 and 3.0 times lower, respectively, compared with the CON group. The MR group had higher ruminal concentrations of short-chain-fatty-acids, propionate, and butyrate on day 56 than the CON group. Adding eubiotics into milk replacer resulted in the highest concentrations of blood insulin-like-growth-factor-I and ß-hydroxybutyrate from days 29 to 56 and the overall experimental period. CONCLUSION: The addition of eubiotic feed additives into the milk replacer can improve health, performance, ruminal fermentation, and biochemical blood indices in dairy calves during the preweaning period.

Potential Novel Biomarkers for Mastitis Diagnosis in Sheep.

This review aims to characterize promising novel markers of ovine mastitis. Mastitis is considered as one of the primary factors for premature culling in dairy sheep and has noticeable financial, productional, and animal welfare-related implications. Furthermore, clinical, and subclinical mammary infections negatively affect milk yield and alter the milk composition, thereby leading to lowered quality of dairy products. It is, therefore, crucial to control and prevent mastitis through proper diagnosis, treatment or culling, and appropriate udder health management particularly at the end of the lactation period. The clinical form of mastitis is characterized by abnormalities in milk and mammary gland tissue alteration or systemic symptoms consequently causing minor diagnostic difficulties. However, to identify ewes with subclinical mastitis, laboratory diagnostics is crucial. Mastitis control is primarily dependent on determining somatic cell count (SCC) and the California Mastitis Test (CMT), which aim to detect the quantity of cells in the milk sample. The other useful diagnostic tool is microbial culture, which complements SCC and CMT. However, all mentioned diagnostic methods have their limitations and therefore novel biomarkers of ovine subclinical mastitis are highly desired. These sensitive indicators include acute-phase proteins, miRNA, and cathelicidins measurements, which could be determined in ovine serum and/or milk and in the future may become useful in early mastitis diagnostics as well as a preventive tool. This may contribute to increased detection of ovine mammary gland inflammation in sheep, especially in subclinical form, and consequently improves milk quality and quantity.

The Association between Selected Dietary Minerals and Mastitis in Dairy Cows-A Review.

The aim of this paper is to describe the association between selected dietary minerals and mastitis in dairy cows. Minerals are a group of nutrients with a proven effect on production and reproductive performance. They also strongly affect immune system function. In particular their deficiencies may result in immunosuppression, which is a predisposing factor for udder inflammation occurrence. The role of selected dietary minerals (including calcium, phosphorus, magnesium, selenium, copper and zinc) has been reviewed. Generally, minerals form structural parts of the body; as cofactors of various enzymes they are involved in nerve signaling, muscle contraction and proper keratosis. Their deficiencies lead to reduced activity of immune cells or malfunction of teat innate defense mechanisms, which in turn promote the development of mastitis. Special attention was also paid to minerals applied as nanoparticles, which in the future may turn out to be an effective tool against animal diseases, including mastitis. To conclude, minerals are an important group of nutrients, which should be taken into account on dairy farms when aiming to achieve high udder health status.

Genomic analysis of European bovine Staphylococcus aureus from clinical versus subclinical mastitis.

Intramammary infections (IMI) with Staphylococcus aureus are a common cause of bovine mastitis and can result in both clinical (CM) or subclinical mastitis (SCM). Although bacterial isolates of S. aureus differ in their virulence potential it is largely unclear which bacterial virulence factors are responsible for increased clinical severity. We performed a genome wide association study and used a generalized linear mixed model to investigate the correlation between gene carriage, lineage and clinical outcome of IMI in a collection of S. aureus isolates from cattle with CM (n = 125) and SCM (n = 151) from 11 European countries. An additional aim was to describe the genetic variation of bovine S. aureus in Europa. The dominant lineages in our collection were clonal complex (CC) 151 (81/276, 29.3%), CC97 (54/276, 19.6%), CC479 (32/276, 11.6%) and CC398 (19/276, 6.9%). Virulence and antimicrobial resistance (AMR) gene carriage was highly associated with CC. Among a selection of nine virulence and AMR genes, CC151, CC479 and CC133 carried more virulence genes than other CCs, and CC398 was associated with AMR gene carriage. Whereas CC151, CC97 were widespread in Europe, CC479, CC398 and CC8 were only found in specific countries. Compared to CC151, CC479 was associated with CM rather than SCM (OR 3.62; 95% CI 1.38-9.50) and the other CCs were not. Multiple genes were associated with CM, but due to the clustering within CC of carriage of these genes, it was not possible to differentiate between the effect of gene carriage and CC on clinical outcome of IMI. Nevertheless, this study demonstrates that characterization of S. aureus CC and virulence genes helps to predict the likelihood of the occurrence of CM following S. aureus IMI and highlights the potential benefit of diagnostics tools to identify S. aureus CC during bovine mastitis.

Effects of various mastitis treatments on the reproductive performance of cows.

BACKGROUND: The purpose of the study described here was to evaluate the effects of different supportive treatments - such as antioxidants, immunomodulators, and nonsteroidal anti-inflammatory drugs (NSAIDs) - in mastitic cows treated with intramammary antibiotics on the efficacy of mastitis therapy and fertility indices. Fertility indices, including time to first insemination, conception rate, time between calving and conception (open days), and number of services per conception (insemination index), were evaluated for 300 dairy cows. Sixty cows without apparent clinical signs of mastitis were assigned 100 days after calving to a Control group. Another 240 cows with clinical mastitis were systematically divided into four experimental groups (I-IV) of 60 cows each. All mastitic cows were treated with approved intramammary antibiotics in recommended doses. Cows in Group I were treated with intramammary antibiotics only. Cows in Groups II, III, and IV, received intramammary antibiotic therapy and a single injection with antioxidants, an immunomodulator (lysozyme dimer), or an NSAID (flunixin meglumine), respectively. RESULTS: The lowest treatment efficacy of mastitic quarters and cows was noted in Group I (51.6 and 53.3%; p > 0.05). The best recovery rate was noted in Group II (63.3 and 66.7%; p > 0.05), followed by Group III (58.3 and 60.9%) and Group IV (58.3 and 58.0%; p > 0.05). The above data did not differ statistically (p > 0.05). The animals with mastitis (Groups I-IV) showed prolonged time to first insemination, more open days, higher insemination index, and lower conception rate than the control cows (p <  0.05). The conception rate of healthy cows and of successfully treated cows was insignificantly lower than that of cows required prolonged antibiotic therapy. Supportive treatments improved the mastitis recovery rate compared with intramammary antibiotics only. The efficacy of mastitis treatments affected the reproduction indices: in cows requiring prolonged treatment with antioxidants, a shorter time to first insemination was needed than in other groups (p <  0.05). Fewer days open were observed between the group with antioxidants and the control group (p <  0.05). CONCLUSIONS: Clinical mastitis negatively affects reproductive indices (days open, pregnancy rate after first AI, NSC) in dairy cows. Different types of supportive medicine, such as antioxidants (vitamin C and E, and ß-carotene), lysozyme dimer, or NSAID can be useful in improving fertility in mastitis cows treated with antibiotic only. It has been proven that each supportive treatment improved antibiotics efficiency and the antibiotic combined with the antioxidants was the most effective treatment.

Non-antibiotic Possibilities in Prevention and Treatment of Calf Diarrhoea.

Due to increasing bacterial antibiotic resistance and the consumers' tendency to choose organic products, cattle farmers are interested in alternative methods of calf diarrhoea treatment. This is a major challenge for veterinarians. Few methods of non-antibiotic treatment that bring satisfactory results have been reported in the related literature so far. In this article, the authors compare different non-antibiotic methods of diarrhoea prevention and treatment in calves. Among the alternatives discussed are herbs, probiotics, prebiotics and synbiotics, lactoferrin, and bacteriophages. It was found that the best results could be achieved through the use of pro-, pre- and synbiotics. However, the authors would like to point out that with the expansion of knowledge about the practical use of broad-scale bacteriophages, they could be the best alternative to antibiotics.