New markers and assays from Porto Conte Ricerche, Sardinia – In addition to the well known problems in dairy cows, mastitis causes significant losses on small ruminant dairy farms. The impact on milk quantity and quality of the subclinical disease is made worse by the suboptimal diagnostic performance of Somatic Cell Counts. In areas where small ruminant farming is an important part of the local economy, such as in Sardinia and in other Mediterranean countries, improving mastitis detection is a primary need. The Research & Development Laboratories at Porto Conte Ricerche, in the heart of the Science and Technology Park of Sardinia, have accepted the daunting challenge of finding new markers and of developing novel, high-performance assays for mastitis.
At the most recent European Mastitis Research Workers meeting in Worcester, England, Filippa Addis explained how, at the R & D Laboratories, they met the challenge. The net results: new findings on immunity of the mammary gland, a high performance biomarker, and new assays that not only improve mastitis detection in small ruminants, but also outperform Somatic Cell Counts for monitoring mastitis in dairy cows.
The challenge: improving mastitis detection in small ruminants
In addition to its relevance for the dairy cow industry, mastitis is also one of the major problems affecting small ruminant production. It is therefore crucial to rely on efficient systems for readily monitoring the appearance of mastitis on all dairy farms, in order to be able to adopt the necessary measures in a short time frame and keep the economic impact to a minimum. This is especially true for subclinical mastitis, due to the difficulties in its detection and therefore to the damage that it can cause to the farmer in terms of reduced quantity and quality of milk production.
In small ruminants, mastitis monitoring is done mainly by measuring the SCC on bulk tank milk as for cows, usually by means of dedicated cell counting instrumentation in a centralised laboratory. When a more stringent control of the flock or herd is needed, SCC is evaluated at the animal or at the half-udder level, once again in the laboratory with cell counting instrumentation or at the farm with the California Mastitis Test (CMT). There is a significant issue, however, which has been the subject of lively debate for several years. In small ruminants the SCC is not as reliable as it is in cows. In fact, there are important limitations due to the influence of numerous physiological, pathological and management variables, such as the extent of epithelial cell sloughing, stage of lactation, parity, milking technique, nutrition, housing, and numerous other factors that can impact SCC quite significantly. As a consequence, the diagnostic performance of the SCC in ewes and goats is not optimal, which is reflected also by the lack of consensus around a defined threshold value. In fact, although the majority of proposed threshold values are lower than 500,000 cells per mL of milk, some authors suggest higher thresholds that can reach up to 1,500,000 cells per mL. In view of all these considerations, a specific effort was clearly needed for developing alternative assays to SCC, having the sensitivity required to detect subclinical mastitis without sacrificing specificity.
Porto Conte Ricerche and the R&D path: applying proteomic technologies for transforming a diagnostic need into a diagnostic tool
The challenge was accepted by the R&D Laboratories of Porto Conte Ricerche, a Regional Government-funded research centre located in Alghero, Sardinia, in view of the outstanding relevance of the problem for the local Sardinian economy. In fact, Sardinia hosts over three million dairy sheep, accounting for 70% of all Italian sheep milk production. Almost all milk is used for cheese making, and the island produces three cheeses with Protected Designation of Origin, namely, Pecorino Sardo, Pecorino Romano and Fiore Sardo.
Porto Conte Ricerche is a research centre of the Science and Technology Park of Sardinia, and its mission is to help local enterprises, especially Small and Medium-sized Enterprises, to remain competitive in an increasingly demanding global market. Therefore, helping farmers and biotech companies in bridging this gap was taken as an important part of its mission. Biomarker discovery and diagnostic development in agrifood, and human and veterinary medicines are among the core research activities of the centre, and its strength leverages on a multidisciplinary environment and on the presence of integrated technological platforms led by researchers with different backgrounds. An invaluable part in facing this challenge was played by different partners actively involved in dairy ruminant mastitis, including the National Reference Centre for Sheep and Goat Mastitis (CRENMOC), the Department of Veterinary Medicine at the University of Milan, and the Regional Farmers’ Association of Sardinia (ARAS).
The strategy of the R&D process was based on the application of a proteomic approach for biomarker identification, followed by the development of immunoassays as the detection strategy. A typical biomarker discovery and diagnostic development pipeline was applied, going through a careful design study, sample analysis by shotgun proteomics, identification of a biomarker panel and large scale validation of the best performing biomarker, followed by the selection of the assay most suited to the diagnostic setting, and finally to its design, construction, and field validation.
The results: novel findings on mastitis and a new, high performance biomarker: cathelicidin
The biomarker discovery path was accompanied by the generation of a vast dataset with information on the identity of milk proteins that change upon mastitis, and the extent of such changes. This provided novel, useful insights on the innate immunity events that take place in the mammary gland upon infection, and on the role that the milk secreting cells play in defending the udder from a detrimental microbial invasion. But the most useful result in terms of mastitis diagnosis was the identification of cathelicidin as a highly promising mastitis marker. Cathelicidin is a small protein that is promptly released in milk in high amounts only when the mammary gland senses the presence of a microbial pathogen, first by epithelial cells and then by polymorphonuclear (PMN) leukocytes. Cathelicidin is a multi-purpose tool of the innate immune response: it has antimicrobial activity, and therefore it kills microbes directly, but it also contributes to the recruitment of large numbers of immune cells into the milk. In consideration of its key role in the immune response process, cathelicidin is perfectly suited as a sensitive and specific mastitis marker that measures inflammation directly and independently from the causative agent. As a further consideration, basing the diagnostic strategy on the detection of proteins causing inflammation, instead of looking for microbial molecules, makes it possible to avoid any unnecessary intervention when the presence of microbes is not causing negative consequences on the animal’s well-being. Actually, as demonstrated by the latest studies on the healthy milk microbiota, the right type of microbial colonization can even be beneficial for udder health.
The test: a milk cathelicidin ELISA for the sensitive and specific detection of mastitis in ewes and cows
ELISA is a widespread, cheap, easy-to-use and high-throughput type of test that does not require dedicated instrumentation or highly trained personnel for carrying out the assay and interpreting the results. It is one of the most popular immunoassay platforms in research and diagnostic laboratories, and it can be quickly and easily implemented in almost any analytical setting. The ability to detect cathelicidin in milk by ELISA opens the way to its simple and straightforward implementation in all analytical laboratories. In addition, on larger dairy farms it might also be feasible to run the cathelicidin ELISA at the farm, as it is currently done with bacterial culture, with a small investment in a plate reader and a little personnel training. This would enable users to implement cathelicidin measurement directly at the farm for a better control of udder health, for following the success of treatment, or for making decisions on selective intramammary infusions with antimicrobials at the dry-off stage.
In the ELISA format, the reliability of cathelicidin as a mastitis marker in ewes has proven excellent. The test shows a good correlation with SCC in full lactation, but its sensitivity is higher. When applying the test to dairy cows, results are even more satisfactory. The cathelicidin ELISA greatly outperforms SCC in terms of sensitivity, and this is without sacrificing specificity. The results have been thoroughly validated, and the details are described in detail in different scientific publications (see below). The cathelicidin ELISA is currently under extensive evaluation for sensitivity and specificity in goats and water buffalos. In ewes and in cows, product development is currently in progress with different commercial partners.
Future perspectives: detection at the farm
The R&D Laboratories at Porto Conte Ricerche are now going further ahead by implementing cathelicidin measurement in other immunoassay formats, with the aim of taking the test outside the laboratory and closer to the farm in the form of portable devices. Usually, these tests do not require complicated procedures, reagents or instruments for carrying out and interpreting test results, and the reaction occurs in a short time. Their main advantage is that the diagnostic information is readily available where it is needed, enabling treatment or culling decisions to be made promptly and without having to lose precious time waiting for test results from the laboratory. The currently available farm test, the CMT, is cheap and provides immediate results, but it is awkward to perform and interpretation is not so straightforward, especially in subclinical mastitis. Last, but not least, it is based on somatic cells, and as such it suffers all of the above mentioned sensitivity and specificity issues of the SCC as measured with cell counters, with the added disadvantage of being even less sensitive. The availability of a high performance and simple portable test would significantly help farmers to control mastitis directly, easily and efficiently and enable the early detection of subclinical mastitis cases, the isolation of affected animals, the quick identification of farm problems and other management issues linked to mastitis. It would also reduce unnecessary interventions including antibiotic administration and ease the implementation of selective intramammary treatment strategies.
Two types of assays suitable for use on the farm are currently under development at Porto Conte Ricerche: the Lateral Flow test (LF) and the Immunobiosensor (IB). The first, the LF is based on the same principle as a pregnancy test: it is a small, cheap, self-enclosed device that does not need reagents or reading instruments and can be easily carried out and interpreted by anyone, without requiring specific skills or training. A prototype is already available and is currently under optimization for providing the highest possible sensitivity. The second, the IB, reproduces the same chemistry as an ELISA test on small magnetic beads but with the important difference that, instead of a coloured product, the enzymatic reaction produces an electric current, which can then be detected with a small screen-printed electrode on a portable reader at the farm. If integrated with an in-line sensor, the IB opens the way to the measurement of cathelicidin in automated milking systems. The IB prototype is currently at an advanced stage of development. Hopefully, both tests will soon be available for commercial implementation by private partners.
Upcoming workshops, training and dissemination events
Porto Conte Ricerche will describe its research and discuss mastitis issues in small and large dairy ruminants together with a selected panel of experts at the following upcoming events:
- Scientific School “Understanding and controlling dairy ruminant mastitis”, Tramariglio, Alghero, 5-8 September 2017;
- 2018 NMC Italy Meeting, Milan, June 11-13, 2018;
- 2018 NMC Satellite Meeting “Focus on Small Ruminant Mastitis”, Tramariglio, Alghero, Sardinia, June 14-15, 2018.
For further information, please visit: portocontericerche.it/en
Scientific articles describing the research and development work Addis M.F., Bronzo, V., Puggioni G.M.G., Cacciotto C., Tedde V., Pagnozzi D., Locatelli C., Casula A., Curone G., Uzzau S., Moroni P. 2017. Relationship between milk cathelicidin abundance and microbiologic culture in clinical mastitis. Journal of Dairy Science. Addis M.F., Tedde V., Puggioni G.M.G., Pisanu S., Casula A., Locatelli C. Rota N., Bronzo, V., Moroni P., Uzzau S. 2016. Evaluation of milk cathelicidin for detection of bovine mastitis. Journal of Dairy Science 99:8250-8258. Addis M.F., Tedde V., Dore S., Pisanu S., Puggioni G.M.G., Roggio A.M., Pagnozzi D., Lollai S., Cannas E.A., Uzzau S. 2016. Evaluation of milk cathelicidin for detection of dairy sheep mastitis. Journal of Dairy Science 99:6446-6456. Addis M.F., Tanca A., Uzzau S., Oikonomou G., Bicalho R.C., Moroni P. 2016. The bovine milk microbiota: insights and perspectives from –omics studies. Molecular BioSystems 12:2359-2372. Addis, M.F., Pisanu, S., Marogna, G., Cubeddu, T., Pagnozzi, D., Cacciotto, C., Campesi, F:, Schianchi, G., Rocca, S., Uzzau, S. 2013. Production and release of immune defense proteins by mammary epithelial cells following Streptococcus uberis infection of sheep. Infection and Immunity 81:3182-3197. Addis, M.F., Pisanu, S., Ghisaura, S., Pagnozzi, D., Marogna, G., Tanca, A., Biosa, G., Cacciotto, C., Alberti, A., Pittau, M., Roggio, T., Uzzau, S. 2011. Proteomics and pathway analysis of the milk fat globule in sheep naturally infected by Mycoplasma agalactiae: insights into the in vivo response of the mammary epithelium to bacterial infection. Infection and Immunity 79:3833-3845.