Minimizing the use of antimicrobials at the end of lactation (dry cow therapy, DCT) requires categorization of cows as likely infected or uninfected. While microbiology is the gold standard for such categorization, the costs of doing so mean that indirect tests such as somatic cell count (SCC) are commonly used. An in-line SCC sensor (SenseHub In Line Somatic Cell Count, in-line SCC) is commercially available but its utility to differentiate cows eligible for dry cow therapy has not been assessed. This prospective diagnostic accuracy study was undertaken to define the sensitivity (Se) and specificity (Sp) of SenseHub SCC against cow-composite milk samples submitted for conventional microbiology. A secondary objective was to assess the utility of SenseHub SCC compared with the maximum (max DHI SCC) or last (last DHI SCC) SCC determined from cow-composite milk samples collected as part of routine herd production recording at monthly intervals throughout lactation. Cows (n = 1,544) from 4 spring-calving, predominantly pasture-fed dairy herds from 3 regions of New Zealand had cow-composite milk samples collected following aseptic teat end preparation immediately before or after the final milking of lactation. These samples were submitted for routine microbiology. The microbiology data from approximately half the cows (n = 770; training data set) were randomly selected after blocking for intramammary infection (IMI) status within herd and these data were used to determine the optimal predictor for indicating IMI from the in-line SCC data by maximizing the area under the receiver operator curve (AUC). The average of the in-line SCC over the final 12 weeks of lactation (in-line 12wSCC) was found to be the best predictor and used for further analyses. The Se and Sp of the in-line SCC for any IMI or for a major pathogen IMI (defined as presence of Staphylococcus aureus, Streptococcus dysgalactiae or Streptococcus uberis) was calculated using the test data set (n = 774). The AUC for the maximum and last DHI SCC were compared with that of the in-line 12wSCC. The cow-level prevalence of any IMI or a major IMI across the entire population was 50.6% and 14.2%, respectively. At a cutpoint of 150,000 cells/mL, Se and Sp of the in-line 12wSCC for any IMI was 0.68 (95%CI 0.64-0.72) and 0.71 (95% CI 0.65-76), respectively, and the Se and Sp for a major pathogen IMI was 0.89 (95%CI 0.82-0.95) and 0.51 (95% CI 0.47-0.55), respectively. The AUC for a major pathogen IMI was 0.82 (95% CI 0.79-0.86), 0.82 (95% CI 0.78-0.86) and 0.84 (95% CI 0.90-0.97) for in-line 12wSCC, max DHI SCC and last DHI SCC, respectively. These AUC did not differ and the AUC for the in-line 12wSCC was non-inferior to that of the last and maximum HT SCC (both P < 0.001). It was concluded that the in-line 12wSCC had an AUC, Se and Sp not different from DHI SCC data and hence this test has utility in selecting cows for different dry cow therapy treatments.

To comply with antibiotic restriction policies in the European Union, internal teat sealants (TS) are increasingly used at drying off (DO) in selective dry cow treatment protocols to maintain udder health. Post-calving TS residue attachment to milking equipment and associated cleaning difficulties is a reason for some farmers to stay away from blanket TS use. Our objective was therefore to improve insight in TS excretion visibility and to compare quantity, pattern, and presence versus absence of TS excretion post-calving between the typical 2 cow categories at DO: High (H) and Low (L) SCC cows, treated with antibiotic (AB) plus TS (H-ABTS) or TS only (L-TS), respectively. In herds in the Netherlands (n = 3), and Germany (n = 4), cows were enrolled at DO, and categorized as H-ABTS (n = 93), or L-TS (n = 99). Post-calving, quarter level TS visibility, quantities, patterns, and percentage of TS infused and excreted post-calving were recorded from 50 mL of pre-milk of every quarter at each of the first 15 or 16 milkings. Udder quarter health status was determined by bacteriological culture and somatic cell counting of quarter milk samples taken at DO and at d 3 post-calving and by clinical mastitis incidence from DO until 30 DIM. Univariable and multivariable models were created to explore associations of TS excretion presence versus absence at the first 3 milkings. Irrespective of SCC category, both laboratory personnel, and farmers saw TS residues at the first milking in an equal 72% of quarters. Compared with laboratory as the gold standard, farmer sensitivity to spot TS in pre-milk was 74.5% at the first milking, decreasing to a maximum of 8.3% at the last 3 milking\'s. At the first milking, TS excretion quantities showed a bimodal distribution pattern and the mean percentage of TS infused (3.83 g) that was excreted in pre-milk at the first milking, was higher in the L-TS (45.5%) compared with the H-ABTS cow category (32%). At the second and third milking, mean adjusted TS percentage excreted was higher in the H-ABTS (8.5% and 1.8%, respectively) compared with the L-TS category (4.6% and 0.4% respectively). The multivariable model of the first 3 milkings showed parity at both the first and second milking, and study group at both the second and third milking, was significantly associated to TS presence. The univariable model showed no association between TS presence at the first milking and udder health. In conclusion, in pre-milk of the first milking, TS residue excretion was bimodal, higher in L-TS cows, more likely present in multiparous cows, and not associated with udder health. At the second and third milking, excretion was higher in H-ABTS cows and TS presence was only more likely in multiparous cows at the second milking.

BACKGROUND: Intramammary infection is the result of invasion and multiplication of microorganisms in the mammary gland and commonly leads to mastitis in dairy animals. Although much has been done to improve cows\' udder health, mastitis remains a significant and costly health issue for dairy farmers, especially if subclinical. In this study, quarter milk samples from clinically healthy cows were harvested to detect pathogens via quantitative PCR (qPCR) and evaluate changes in individual milk traits according to the number of quarters infected and the type of microorganism(s). A commercial qPCR kit was used for detection of Mycoplasma bovis, Mycoplasma spp., Staphylococcus aureus, coagulase-negative staphylococci (CNS), Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Prototheca spp., Escherichia coli, Klebsiella spp., Enterococcus spp. and Lactococcus lactis ssp. lactis. Quarter and pooled milk information of 383 Holstein, 132 Simmental, 129 Rendena, and 112 Jersey cows in 9 Italian single-breed herds was available.
RESULTS: Among the cows with pathogen(s) present in at least 1 quarter, CNS was the most commonly detected DNA, followed by Streptococcus uberis, Mycoplasma bovis, and Streptococcus agalactiae. Cows negative to qPCR were 206 and had the lowest milk somatic cell count. Viceversa, cows with DNA isolated in ≥ 3 quarters were those with the highest somatic cell count. Moreover, when major pathogens were isolated in ≥ 3 quarters, milk had the lowest casein index and lactose content. In animals with pathogen(s) DNA isolated, the extent with whom milk yield and major solids were impaired did not significantly differ between major and minor pathogens.
CONCLUSIONS: The effect of the number of affected quarters on the pool milk quality traits was investigated in clinically healthy cows using a commercial kit. Results remark the important negative effect of subclinical udder inflammations on milk yield and quality, but more efforts should be made to investigate the presence of untargeted microorganisms, as they may be potentially dangerous for cows. For a smarter use of antimicrobials, analysis of milk via qPCR is advisable - especially in cows at dry off - to identify quarters at high risk of inflammation and thus apply a targeted/tailored treatment.

The objective of this study was to determine quarters requiring antimicrobial treatment using either a benchtop somatic cell counter or culture with gram-positive selective media and compare the outcomes in these cows to those receiving blanket dry cow therapy (BDCT) in a randomized, controlled trial. We evaluated 2 novel methods of identifying cows with intramammary infections followed by selective antimicrobial treatment at a commercial dairy farm to determine their usefulness in decreasing antibiotic usage during the dry period without significant detrimental effects on milk quality and production. Cows (n = 840) were randomly allocated to one of 3 groups (BDCT, gram-positive selective media culture-based selective dry cow therapy [C-SDCT], and somatic cell count-based SDCT [S-SDCT]) the day before dry-off, and quarter-level milk samples (QLMS) were collected. The QLMS from cows in the S-SDCT group were evaluated using the cell counter, and quarters were treated if SCC was ≥200,000 cells/mL, whereas the QLMS from cows in the C-SDCT group were cultured, and quarters were treated if the culture showed growth. All cows in the BDCT received antimicrobial therapy, and all cows received an internal teat sealant regardless of treatment group. Outcomes measured were first and second DHIA test SCC, milk production through 60 DIM, cows leaving the farm, clinical mastitis, and bacteriologic new infections in a subset of quarters. Cows in both SDCT groups had fewer antimicrobial treatments than cows in the BDCT group as was expected, and cows in the C-SDCT group had fewer treatments than those in the S-SDCT group. Cows in both SDCT groups had a higher linear score at the first DHIA test (BDCT: 1.8, S-SDCT: 2.2, C-SDCT: 2.2); however, we found no other differences between groups regarding any other outcomes measured. Although antimicrobial use was significantly reduced, farms should use caution in adopting the benchtop analyzer and the selective media described in this study as ways to identify infected cows for dry cow therapy because they may result in increased linear scores early in lactation.

Mycoplasma bovis has recently been identified increasingly in dairy cows causing huge economic losses to the dairy industry. M. bovis is a causative agent for mastitis, pneumonia, endometritis, endocarditis, arthritis, otitis media, and many other clinical symptoms in cattle. However, some infected cows are asymptomatic or may not shed the pathogen for weeks to years. This characteristic of M. bovis, along with the lack of adequate testing and identification methods in many parts of the world until recently, has allowed the M. bovis to be largely undetected despite its increased prevalence in dairy farms. Due to growing levels of antimicrobial resistance among wild-type M. bovis isolates and lack of cell walls in mycoplasmas that enable them to be intrinsically resistant to beta-lactam antibiotics that are widely used in dairy farms, there is no effective treatment for M. bovis mastitis. Similarly, there is no commercially available effective vaccine for M. bovis mastitis. The major constraint to developing effective intervention tools is limited knowledge of the virulence factors and mechanisms of the pathogenesis of M. bovis mastitis. There is lack of quick and reliable diagnostic methods with high specificity and sensitivity for M. bovis. This review is a summary of the current state of knowledge of the virulence factors, pathogenesis, clinical manifestations, diagnosis, and control of M. bovis mastitis in dairy cows.

The study of new indirect methods for mastitis detection is of great relevance both at the economic level of the farm and dairies, and in terms of consumer health, and animal welfare. These methods help us to monitor the disease and speed up the decision-making process on treatment of the affected animal and the destination of the milk. The main aim of this work was to study the effect of intramammary infection and other non-infectious factors on the activity of the enzyme N-acetyl-β-D-glucosaminidase (NAGase) in milk, in order to evaluate its use as an indicator for the early diagnosis of mastitis in sheep that could be less expensive, easier to measure and a better marker of inflammation or complementary to existing methods such as somatic cell count (SCC). Seven biweekly samplings were carried out, in which NAGase activity, SCC and milk were analyzed. Glands were classified according to their sanitary status based on the results of the SCC and bacteriological analysis. Non-infectious factors such as lactation stage, parity number and milking session had a statistically significant effect on NAGase values, finding the highest NAGase values at the onset and end of the study, in infectious mastitic glands of multiparous females and at morning milking. However, among the NAGase variation factors studied, the health status of the gland was the factor that caused the highest variation in enzyme levels, with infectious mastitic glands showing higher values than healthy glands. The predictive ability of NAGase was also studied by means of several logistic regression models, with the one that included NAGase together with lactation stage and parity obtaining the best results if sensitivity is to be prioritized, or the model that included NAGase, lactation stage, parity, milking and production if specificity is to be prioritized. From the results obtained, it can be concluded that the use of NAGase as an intramammary infection detection method in sheep can be useful when non-infectious factors that cause changes in the concentration of the enzyme are also considered.

Mastitis is a major health problem for bovines and can be categorized as non-severe or severe, based on clinical symptoms. A severe case of clinical mastitis is usually defined by the cow being affected systemically. It is important to consider how to handle severe cases because these cases can be fatal and cause high production losses. However, there are generally few detailed treatment guidelines. By conducting a scoping review on the topic, we aimed to synthesize the information that is available on treatment and outcomes, as reported from clinical trials and observational studies. This was facilitated by following the PRISMA-guidelines with a stepwise systematic screening of scientific literature on the subject, retrieved via Pubmed and Web of Science, using pre-defined selection criteria. The results yielded a total of 14 reports of treatment and outcomes in cases of naturally occurring severe clinical mastitis. Cross-trial comparison was difficult due to the different exclusion criteria and outcome definitions. Many studies focused on cases caused by gram-negative bacteria treated with intensive antibiotic protocols, often containing antibiotics that are categorized as critical for human health. Few focused on severe cases caused by gram-positive bacteria or on the relative use of non-antibiotic treatment. In general, only a small number of statistically significant differences were found in trials comparing different treatment protocols, with no obvious trends across trials. Our findings emphasize the need for more research into the treatment efficacy of antibiotic and non-antibiotic options for clinically severe mastitis. Furthermore, consideration of how trial conditions relate to the practical circumstances in a field setting could improve the applicability of reported results. This could help to provide practitioners with the information needed to make evidence-based treatment decisions in cases of clinically severe mastitis.

Streptococcus uberis is one of the primary causative agents of mastitis, a clinically and economically significant disease that affects dairy cattle worldwide. In this study, we analyzed 140 S. uberis strains isolated from mastitis milk samples collected from 74 cow herds in the Czech Republic. We employed whole-genome sequencing to screen for the presence of antimicrobial resistance (AMR) genes and genes encoding virulence factors, and to assess their genetic relationships. Our analysis revealed the presence of 88 different sequence types (STs), with 41% of the isolates assigned to global clonal complexes (GCCs), the majority of which were affiliated with GCC5. The STs identified were distributed across the major phylogenetic branches of all currently known STs. We identified fifty-one putative virulence factor genes, and the majority of isolates carried between 27 and 29 of these genes. A tendency of virulence factors and AMR genes to cluster with specific STs was observed, although such clustering was not evident within GCCs. Principal component analysis did not reveal significant diversity among isolates when grouped by GCC or ST prevalence. The substantial genomic diversity and the wide array of virulence factors found in S. uberis strains present a challenge for the implementation of effective anti-mastitis measures.

The low susceptibility to mastitis of female donkey (jenny) mammary glands and the strong immune properties of donkey milk are acknowledged, but little is known about the genes involved in mammary gland immunity in jennies. Herein, we used RNA-sequencing and bioinformatics analyses to explore jenny mammary gland transcriptomes and detect potential functional differentially expressed (DE) mRNAs related to immunity during four specific developmental stages: foetal (F), pubertal (P), adult parous nonlactation (N) and lactation (L). A total of 2497, 583 and 1820 DE mRNAs were identified in jenny mammary glands at F vs. P, P vs. N, and N vs. L, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) analyses revealed numerous GO terms related to immune function, especially between F and P. Seven significantly enriched profiles were identified, among which 497 and 1261 DE mRNAs were upregulated in profiles 19 and 17. Eleven mRNAs were enriched in over 10 KEGG pathways. β-2-microglobulin (B2M), immunoglobulin heavy constant mu (IGHM), toll like receptor 2 (TLR2), toll like receptor 4 (TLR4) and myeloid differentiation factor 88 (MYD88) were mainly involved in phosphoinositide 3-kinase (PI3K)-Akt signalling, phagosome and nuclear factor kappa-B (NF-kappa B) signalling pathways. The findings provide insight into the molecular features underpinning the low prevalence of intramammary infections (i.e., mastitis) in donkeys.

Staphylococcus aureus is one of the agents of bovine mastitis of hardest control due to a complex pathogenesis comprising a variety of virulence factors, which ensures its persistence in the mammary gland, causing significant health and economic losses. Therefore, understanding the pathogenesis of this agent is imperative. Galleria mellonella has stood out as an invertebrate animal model for the study of infectious diseases that affect several hosts. This work aimed to evaluate G. mellonella larvae as an experimental model for the study of virulence phenotypes in an S. aureus population isolated from bovine mastitis. Thirty genetically divergent S. aureus strains were chosen based on PFGE analysis. After experimental infection, larvae survival rates, bacterial growth in hemolymph, melanization intensity of the dorsal vessel, and histological characteristics of the infected tissues were evaluated. The G. mellonella model showed a clear diversity in the S. aureus pathogenicity pattern, allowing the differentiation of strains with virulence phenotypes ranging from high to low degrees. Histological analysis confirmed that the strains tested were capable of inducing the formation of nodules and melanization spots in the dorsal vessels of the larvae in different magnitudes. The strains 16S-717, 19C-828, and 31S-1443 presented the highest virulence intensity among the bacteria tested and will be used further for the generation of S. aureus mutant populations to prospect genetic targets aimed to develop control strategies of bovine mastitis. Altogether, our results suggest that G. mellonella is an attractive and low-cost animal model for characterizing virulence phenotypes of large S. aureus populations.