Biofilm formation is of growing concern in human and animal health. However, it is still unclear how biofilms are related to mastitis infections in dairy cattle. In this study, a comparison between two tests for biofilm formation and the association between biofilm and the presence of genes associated with biofilm formation were investigated for 92 Staphylococcus aureus isolates from intramammary infections. Congo red agar (CRA) and microtitre test assay (MTA) in vitro phenotypic tests were used to evaluate biofilm formation. The presence of icaA, icaD, and bap genes associated with biofilm formation was confirmed using the polymerase chain reaction. Results show that most of the S. aureus isolates, though not possessing one of the biofilm-forming genes, were able to produce biofilms. MTA was more frequently positive in identifying biofilm-forming isolates than CRA.

The mode of action of bismuth subnitrate in teat sealant formulations as a preventative for intramammary infections during the dry period is unknown. Although previous studies proposed an action mechanism-creating a physical barrier in the teat canal to prevent bacterial invasion-it has not been proven experimentally. We hypothesized that bismuth subnitrate has an inhibitory effect on bacterial growth, in addition to its barrier effect. The objective of this study was to assess the effect of bismuth subnitrate on bacterial growth of major mastitis-causing agents. A strain of Streptococcus uberis (SR115), 2 strains of Staphylococcus aureus (SA3971/59 and SA1), and a strain of Escherichia coli (P17.14291) were tested in vitro for their ability to grow in the presence or absence of bismuth subnitrate. Disk diffusion testing, impedance measurement, and evaluation of bacterial growth in shaking conditions were the methods used to test this hypothesis. A reduction of growth in the presence of bismuth subnitrate occurred for all the strains tested. However, we observed strain and species variations in the extent of growth inhibition. These results suggest that an inhibitory effect on bacterial growth by bismuth subnitrate could partially explain the efficacy of bismuth-based formulations for preventing intramammary infections over the dry period. Further research is required to test the effect of teat sealant formulations on bacterial growth.

Experimental challenge models can be used to compare infectivity and pathogenicity of different strains of mastitis-causing bacteria, the efficacy of pharmaceutical products and host-responses in a cost-effective manner.

New Zealand Dairy cows are predominantly milked twice a day (TAD), however full-season once a day (OAD) milking was practiced by ~9% of herds in New Zealand in the season 2015-2016 (Edwards, 2018). OAD milking offers benefits to both the cow and farmer and has been detailed previously by Stelwagen et al. (2013). Changing from TAD to OAD causes a reduction in milk volume and introduces changes in milk composition. A positive effect of OAD is a significant increase in the content of valuable bioactive molecules, such as lactoferrin (LF), and other immune-related proteins in milk, such as acute phase proteins and RNases (Farr et al., 2002; Stelwagen et al., 2011). LF is produced in bovine milk as part of a wide range of bioactive proteins and peptides, which have roles in immunity and immunoregulation functions (Galfi et al., 2016). LF has been found to have antibacterial activity for bacteria with high iron requirements like Escherichia coli and Staphylococcus aureus, coagulase negative Staphylococcus sp. and Pseudomonas aeruginosa (González-Chávez, Arévalo-Gallegos, & Rascón-Cruz, 2009). In addition, LF exhibits antiviral activity to both DNA and RNA viruses, and antifungal activity against Candida sp., Aspergillus sp. and Trichophyton sp. (González-Chávez et al., 2009). Lactoferrin has been investigated for the last 20 years for its ability to stimulate immune response by inducing granulopoiesis, participating in the process of cytokines and antibodies synthesis in vitro, activating complement and producing interleukins (IL-1 & IL-2) and tumor necrosis factor D (Legrand, 2011). In addition, LF was characterized as a factor intervening in the fight of cancer (Duarte, Nicolau,

The aim of this study was to assess the effect of bismuth subnitrate and micronized keratin on bovine neutrophils in vitro. We hypothesized that recruitment and activation of neutrophils into the teat canal and sinus are the mechanisms of action of bismuth subnitrate and keratin-based teat sealant formulations. To test this, a chemotaxis assay (Experiment 1) and a myeloperoxidase (MPO) assay (Experiment 2) were conducted in vitro. Blood was sampled from 12 mid-lactation dairy cows of variable ages. Neutrophils were extracted and diluted to obtain cell suspensions of approximately 106 cells/mL. In Experiment 1, test substances were placed in a 96-well plate, separated from the cell suspension by a 3 µm pore membrane and incubated for 3 h to allow neutrophils to migrate through the membrane. In Experiment 2, neutrophils were exposed to the test products and the amount of MPO released was measured by optical density. Results showed that neutrophils were not activated by bismuth or keratin products (p < 0.05) in all of the tests performed. These results suggest that the mechanisms of action of bismuth subnitrate and keratin-based teat sealants do not rely on neutrophil recruitment and activation in the teat canal and sinus after treatment.