INTRAMAMMARY P RECISE PRECISE QUARTER-LEVEL ESTIMATION OF THE IMPACT OF QUARTER NON-AUREUS STAPHYLOCOCCAL INTRAMAMMARY INFECTION ON UDDER HEALTH AND MILK YIELD IN DAIRY HEIFERS INFECTION - LEVEL DIMITRI VALCKENIER ESTIMATION ON UDDER OF HEALTH THE IM P AND ACT MILK OF NON YIELD - AUREUS IN DAIRY STA HEIFERS P HYLOCOCCAL D IMITRI V ALCKENIER 2021 Precise Quarter-Level Estimation of the Impact of Non-Aureus Staphylococcal Intramammary Infection on Udder Health and Milk Yield in Dairy Heifers Dimitri Valckenier Merelbeke, 2021 “A pessimist sees difficulty in every opportunity; an optimist sees opportunity in every difficulty.” Winston Churchill “The pessimist complains about the wind; the optimist expects it to change; the realist adjusts the sails.” William Arthur Ward Precise Quarter-Level Estimation of the Impact of Non-Aureus Staphylococcal Intramammary Infection on Udder Health and Milk Yield in Dairy Heifers Dimitri Valckenier Cover design: Creativision, Erpe-Mere Printing: University Press, Wachtebeke ISBN number: 9789464202434 Department of Reproduction, Obstetrics, and Herd Health Faculty of Veterinary Medicine Ghent University Precise Quarter-Level Estimation of the Impact of Non-Aureus Staphylococcal Intramammary Infection on Udder Health and Milk Yield in Dairy Heifers Dimitri Valckenier Dissertation submitted to Ghent University in the fulfillment of the requirements for the degree of Doctor in Veterinary Sciences (PhD) June 18, 2021 Promotors Prof. dr. Sarne De Vliegher Faculty of Veterinary Medicine, Ghent University, Belgium Prof. dr. Sofie Piepers Faculty of Veterinary Medicine, Ghent University, Belgium Members of the Examination Committee Prof. dr. Edwin Claerebout, chairman Faculty of Veterinary Medicine, Ghent University, Belgium Dr. Anneleen De Visscher Flanders Research Institute for Agriculture, Fisheries, and Food (ILVO), Belgium Prof. dr. Marie Joossens Faculty of Sciences, Ghent University, Belgium Prof. dr. Gerrit Koop Department Population Health Sciences, Utrecht University, the Netherlands Prof. dr. Bart Pardon Faculty of Veterinary Medicine, Ghent University, Belgium Prof. dr. Ynte H. Schukken GD Animal Health, the Netherlands – Department of Animal Sciences, Wageningen University, the Netherlands – Department of Population Health Sciences, Utrecht University, the Netherlands Table of Contents Chapter 1 General introduction 1 Chapter 2 Scope and aims of the thesis 29 Chapter 3 Effect of intramammary infection with non-aureus staphylococci 33 in early lactation in dairy heifers on quarter somatic cell count and quarter milk yield during the first 4 months of lactation Chapter 4 The effect of intramammary infection in early lactation with non- 65 aureus staphylococci in general and Staphylococcus chromogenes specifically on quarter milk somatic cell count and quarter milk yield Chapter 5 Longitudinal study on the effects of intramammary infection with 103 non-aureus staphylococci on udder health and milk production in dairy heifers Chapter 6 General discussion 143 Summary 191 Samenvatting 197 Curriculum vitae and publications 205 Dankwoord 213 List of Abbreviations AMS Automatic milking system AS Ali and Schaeffer BMSCC Bulk milk somatic cell count CFU Colony forming units cIMI Cured intramammary infection CM Clinical mastitis d Days DCC Direct cell counter DHI Dairy herd improvement DHIA Dairy Herd Improvement Association DIM Days in milk IMI Intramammary infection LnSCC Natural log-transformed somatic cell count LSM Least squared means MALDI-TOF Matrix-assisted laser desorption/ionization time of flight MLST Multilocus sequence typing Mo Months MS Mass spectrometry MY Milk yield NAGase N-acetyl-β-D-glucosaminidase NAS Non-aureus staphylococci nIMI New intramammary infection PCR Polymerase chain reaction PFGE Pulsed-field gel electrophoresis pIMI Persistent intramammary infection PMNL Polymorphonuclear leukocytes PRL Prolactin q Quarter qMY Quarter milk yield qPRL Quarter milk prolactin qSCC Quarter milk somatic cell count RAPD-PCR Random amplification of polymorphic DNA - polymerase chain reaction RIA Radioimmunoassay S. Staphylococcus SCC Somatic cell count SCM Subclinical mastitis SD Sampling day SE Standard error tDNA-PCR Transfer RNA intergenic spacer - polymerase chain reaction tIMI Transient intramammary infection Chapter 1 General introduction D. Valckenier Department of Reproduction, Obstetrics, and Herd Health Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium Chapter 1 General introduction 1. Bovine mastitis 1.1. What is bovine mastitis? Bovine mastitis, defined as an inflammation of the mammary gland parenchyma (Fig. 1), is an immunological reaction to infectious or noninfectious injury (e.g., physical trauma or toxic agents). The majority of mastitis cases are associated with bacteria, with over 137 species, subspecies and serovars that have been isolated from the mammary gland (Watts, 1988). A minority of mastitis cases can be attributed to other organisms such as yeasts, fungi, algae (Watts, 1988) and viruses (Gourlay et al., 1974; Wellenberg et al., 2002; Çomakli and Özdemir, 2019). Intramammary infections (IMI) are usually caused by bacteria invading the bovine mammary gland through the teat canal by growth or propulsion (Blowey and Edmondson, 2010) and subsequently adhering to the mammary tissue. Based on the clinical presentation, mastitis can be categorized as subclinical mastitis (SCM) or clinical mastitis (CM). In cases of SCM there are no visible changes to the milk, udder, and cow (Ruegg, 2011); however, there is an inflammatory reaction resulting in an influx of white blood cells, primarily polymorphonuclear neutrophil leukocytes. The most common method to detect this type of mastitis is by measuring the somatic cell count (SCC) in the milk. Inflammation in the mammary gland combined with the potential adverse effects of invading bacterial pathogens in the udder parenchyma can result in suboptimal milk production in the affected quarter (Koldeweij et al., 1999; Seegers et al., 2003; Halasa et al., 2007). Conversely, in cases of CM, a variety of symptoms can be observed such as altered milk composition (e.g., flakes, watery milk, …), local symptoms in the udder (e.g., swelling, loss of the function of a quarter, …), systemic symptoms, and even death (Ruegg, 2011). 3 Chapter 1 General introduction Figure 1: Schematic illustration of the anatomy of one udder quarter from the bovine mammary gland. Adapted from http://www.ubrocare.com/content/udder-anatomy 1.2. Importance of bovine mastitis in general and heifer mastitis in specific After more than 30 years, the dairy sector in the European Union entered a new economic context in 2015 with the termination of the milk quota system. As milk prices become more volatile and resources for milk production (e.g., nutrition, ground, labor, ...) more expensive, optimizing the milk production and management on dairy farms is more crucial than ever. Additionally, an increasing trend in herd size and specialization in the dairy industry requires ever-increasing investments of the herd owner. The income of most dairy farms relies almost entirely on the selling of raw milk; therefore, prevention of diseases that affect dairy cow milk 4 Chapter 1 General introduction production is of the utmost importance. Producing large quantities of milk requires a well- developed and healthy udder to meet the quality standards. For example, infection of the udder tissue in the developing udder (Trinidad et al., 1990a) or during the first lactation (De Vliegher et al., 2005b) could potentially threaten the milk production capacity of the developing udder and might impair the udder health for the duration of the productive life of the dairy cow (Rupp et al., 2000). Heifers, i.e., cows in their first lactation, form the future of each dairy herd. They replace older cows at the end of their productive life, both for milking and breeding. On well-managed dairy farms, implementation of continuous genetic selection and well-considered breeding programs results in new generations of animals with higher genetic merit for milk production than previous generations. Therefore, the goal of each dairy herd should be to raise bred heifers in optimal conditions to maximize the expression of their genetic potential. The IMI status of pre-calving heifers is seldomly checked because pre-existing IMI before calving is unexpected (Nickerson, 2009) and it is less feasible to collect mammary secretion samples. Unfortunately, heifers often have IMI before their first calving. Depending on the herd and region, estimated IMI prevalences range from 12% to 75% of the quarters of heifers before and around calving (Oliver and Mitchell, 1983; Trinidad et al., 1990b; Roberson et al., 1994; Parker et al., 2007; Fox, 2009). Infections of the udder in late gestation or in lactation form a threat to the genetic potential of heifers (De Vliegher et al., 2012). The impact of IMI depends on, amongst others, the form of mastitis (subclinical versus clinical), the pathogenicity and virulence of the invading bacteria (major versus minor pathogens), the time and duration of the infection relative to calving, and the host’s immunity (De Vliegher et al., 2012). Heifer mastitis results in extra costs and/or financial losses that can be ascribed to milk production losses, additional labor, use of drugs, veterinary needs, premature culling, production of nonsaleable milk and risk of residues. The combined costs of an elevated SCC in early lactation that decreased again, an elevated