1The aim of this thesis is to increase our understanding of the role of polymorphisms in the bovine CXCR1 (IL8RA) gene in mammary gland immunity and udder health. Associations between CXCR1 polymorphisms and neutrophil viability, subclinical mastitis, clinical mastitis and milk yield were detected indicating CXCR1 to be a major gene for mastitis resistance.
Neutrophils play a major role in the mammary gland immunity. Binding of interleukin-8 (IL-8) on chemokine (C-X-C motif) receptor 1 (CXCR1) causes migration from blood to the site of infection and enhances activity and viability of neutrophils. Despite its important function, we have detected 16 polymorphisms in the coding region of CXCR1 potentially affecting IL-8 binding or signal transduction.
In a first study, associations between CXCR1 polymorphisms and viability and concentration of milk neutrophils of 73 early lactating dairy heifers were studied. Resident milk neutrophils showed a lower percentage apoptosis in c.980AG heifers compared to c.980GG heifers. Additional to viability, activity of neutrophils might associate with CXCR1 polymorphisms. To test this hypothesis, reactive oxygen species (ROS) generation of blood neutrophils isolated from twenty early lactating heifers with different CXCR1 genotype was studied. Genotype was neither associated with ROS generation in general nor with the effect of IL-8 incubation on reactive oxygen species generation.
In a third study, associations between CXCR1 polymorphisms and subclinical heifer mastitis of 140 heifers belonging to 20 commercial dairy herds were analyzed. Polymorphism c.980A>G was associated with pathogen-group specific intramammary infections; and heifers with genotype c.980AG were less likely to be infected with major mastitis pathogens compared to heifers with genotype c.980GG.
To further study the influence of polymorphism c.980A>G on mammary gland immunity, a fourth study was conducted in which quarters of 4 c.980AG and 4 c.980GG Holstein heifers were experimentally infected with a Staphylococcus chromogenes isolate. Both genotype groups rapidly eliminated the bacteria. Yet, c.980AG heifers showed a lower somatic cell count and milk neutrophil viability during the early immune response compared to c.980GG heifers.
Unlike other regions, little information on clinical mastitis was available in Flanders. Before testing associations between clinical mastitis and CXCR1 genotype, pathogen-specific incidence rate of clinical mastitis (IRCM) was estimated on 50 randomly selected commercial Flemish dairy herds. During a one-year survey, participating producers sampled 845 cases and 692 dairy cows. The median IRCM was estimated at 5.3 quarter cases per 10,000 cow-days at risk. Streptococcus uberis and Escherichia coli were the most frequently isolated pathogens. Overall IRCM and E. coli IRCM were higher in dirty compared to clean herds based on udder hygiene scores. In total, 3,106 cows from the above-mentioned survey were genotyped for polymorphisms c.735C>G and c.980A>G. Cows with genotype c.735GG had lower Staphylococcus aureus IRCM and produced on average 0.8 kg milk more per day compared to cows with genotype c.735CC. Additionally, a parity-specific association between Staphylococcus aureus IRCM and SNP c.980A>G was detected. Heifers with genotype c.980GG had a lower Staphylococcus aureus IRCM compared to heifers with genotype c.980AG.
In the final study, gene expression was measured in milk somatic cells isolated from the experimentally infected heifers. The experimental infection but not polymorphism c.980A>G influenced expression of CXCR1 and many of commonly used reference genes.