Lactoferrin in Bovine Intramammary Infection

Abstract

Lactoferrin (Lf) is a bioactive molecule found in milk that is expressed in and secreted by mammary epithelial cells. It is also present in the secondary granules of neutrophils. The concentration of Lf in milk depends on the stage of lactation of the cow and infection status of the mammary gland. Lf has many biological functions that relate to bovine intramammary infections. Lf exerts bacteriostatic activity by binding iron and has bactericidal activity that results from the interaction between Lf and bacterium. Lf regulates the inflammatory response by binding to bacterial endotoxin, which inhibits formation of pro-inflammatory cytokines, or indirectly stimulates the host immune system. Lf inhibits intracellular invasion of pathogens and biofilm formation. It has immunoregulatory functions that affect antibody synthesis, production of various cytokines, and complement activation.

Mastitis is the most costly disease of dairy cows, causing decrease in milk quality and safety and marked economic losses for the industry. Mastitis affects animal welfare and is one of the main reasons for culling dairy cows. Mastitis is the most common reason for treating dairy cows with antibiotics, but results of the treatments are often poor. New approaches for mastitis control are urgently needed.

This thesis focuses on the behavior and effects of Lf in the bovine mammary gland. The studies include in vivo experiments, using induced mastitis with endotoxin, and two different udder pathogens, as well as in vitro experiments using bovine mammary epithelial (BME) cells. Concentrations of bovine Lf (bLf) and citrate in milk were investigated in experimentally induced endotoxin mastitis using the same cows during early and late-lactation period. The ability of coagulase-negative staphylococci (CNS) isolated from bovine mastitis to adhere to, and invade and replicate intracellularly, and the related effect of bLf, were studied in a BME cell model. Transgenic cows expressing recombinant human Lf (rhLf) in their milk were used in two experiments in which intramammary infection was induced by a major pathogen Escherichia coli, and a minor pathogen, Staphylococcus chromogenes. Basal concentrations of bLf and rhLf in the milk of the rhLf-transgenic cows were determined during the first months of the lactation period.

Concentration of bLf in the milk was significantly higher during the early lactation period than during the late lactation in experimentally induced endotoxin mastitis. The molar ratio of citrate to bLf before and after challenge was also significantly higher during the early lactation period. The molar ratio is more important than the absolute concentration of either component, and a low molar ratio increases resistance of the mammary gland to invading bacteria, coliforms in particular. The results of this study may partly explain the susceptibility of dairy cows to intramammary infections during early lactation.

All CNS species examined were able to adhere to BME cells, but internalization varied among the strains. The effect of bLf on the adhesion and invasion of the CNS strains was weak, but it significantly decreased intracellular replication rates. The effect of bLf was not related to the in vitro susceptibility of the CNS strain to bLf.

A new approach to prevention of bovine intramammary infection could be represented by genetic engineering, which was tested here using rhLf-transgenic cows. Two different udder pathogens, E. coli and S. chromogenes, were selected for induction of experimental mastitis in the transgenic and control cows. The bacterial strains used in the experiments were susceptible in vitro to bLf; susceptibility to hLf varied such that the E. coli strain was intermediately susceptible and the S. chromogenes strain fully susceptible. The response of the cows was monitored by determining concentrations of bLf and rhLf in the milk and concentrations of several indicators of inflammation in the milk and blood. Expression of rhLf in the milk did not protect the cows from E. coli or staphylococcal intramammary infection. All cows became infected in both models. In the E. coli model all cows developed clinical mastitis, but rhLf-cows showed milder systemic signs and lower concentrations of cortisol and haptoglobin in serum than the controls. rhLf-cows were protected from clinical disease in staphylococcal infection and had a milder inflammatory reaction, which was recorded as lower N-acetyl-β-D-glucosamidase (NAGase) activity and concentration of serum amyloid A in the milk.

The results of the investigations reported in this thesis show that one aspect of the innate immunity of the mammary gland is mediated by Lf, which has a multifunctional role. The host response of the cow to intramammary infection depends on the stage of lactation, and on the infectious agent and its virulence. The limited positive effects in the experiments using rhLf-transgenic cows do not warrant further development of this type of genetic engineering to increase mastitis resistance in dairy cows. However, Lf could be of potential benefit if it were able to be used to support the immune defense of the bovine mammary udder.

Laktoferriini, yksi maidon bioaktiivisista proteiineista, on osa utareen luonnollista puolustusjärjestelmää. Maidon laktoferriinipitoisuus on korkeimmillaan poikimisen jälkeen ja umpeenmenovaiheessa. Laktoferriinpitoisuus nousee utaretulehduksessa eli mastiitissa. Laktoferriinin vaikutus utaretulehduksessa on epäspesifinen ja moninainen. Se mm. estää bakteerien kasvua sitomalla vapaata rautaa ja heikentää bakteerien tarttumista epiteelisoluihin sekä edelleen soluinvaasiota tai biofilmin muodostamista solun pinnalle. Laktoferriini säätelee myös immuunivasteen voimakkuutta gram-negatiivisten bakteerin aiheuttamissa infektioissa sitoutumalla ulkokalvon endotoksiiniin, heikentäen sen vaikutusta ja samalla vahingoittaen bakteerisolua.

Tässä väitöskirjatyössä tarkasteltiin (1) mahdollisuutta parantaa lehmän maitorauhasen vastustuskykyä infektioille lisäämällä maidon laktoferriinipitoisuutta geeniteknologian avulla, (2) laktaatiokauden vaikutusta tulehduksen voimakkuuteen ja maidon laktoferriini- ja sitraattipitoisuuksiin kokeellisessa endotoksiinimastiitissa ja (3) laktoferriinin vaikutusta koagulaasi-negatiivisten stafylokokkien (KNS) adheesioon, invaasioon ja solunsisäiseen lisääntymiseen utare-epiteelisolumallissa. Transgeenisten lehmien maitorauhasessa ilmentyvän ja maitoon erittyvän humaanilaktoferriinin pitoisuus oli kymmenkertainen verrattuna normaalin lehmän maidon laktoferriinipitoisuuteen. Kokeelliset tartutuskokeet transgeenisillä ja kontrollilehmillä tehtiin kahdella erityyppisellä utaretulehdusta aiheuttavalla bakteerilla, Escherichia coli ja KNS. Ylimääräinen humaanilaktoferriini maidossa ei suojellut lehmiä kummaltakaan tartunnalta. E. coli –mallissa kaikki lehmät sairastuivat, mutta transgeenisten lehmien oireet olivat lievempiä kuin normaaleilla lehmillä. Stafylokokkimallissa erot transgeenisten ja kontrollilehmien välillä olivat selvemmät: transgeenisistä lehmistä yksikään ei saanut näkyvää utaretulehdusta ja tulehdusreaktiot olivat lievemmät kuin verrokkilehmillä.

Kokeellinen endotoksiinimastiitti laktaatiokauden alussa ja lopussa samoilla lehmillä osoitti, että maidon sitraatin molaarinen suhde laktoferriiniin oli lypsykauden alussa merkitsevästi korkeampi kuin lypsykauden lopussa. Havaittu ero lienee ainakin osittain yhteydessä lehmien utaretulehdusherkkyyteen poikimisen jälkeen. Utare-epiteelisolumallissa laktoferriini vähensi merkittävästi stafylokokkien kykyä lisääntyä solunsisäisesti, kun taas vaikutus adheesioon ja invaasioon oli heikko.

Tutkimustulokset osoittavat, että laktoferriinillä on vaikutusta lehmän luontaiseen vastustuskykyyn utaretulehduksessa ja että maidon laktoferriinipitoisuuteen sekä utaretulehdukseen vaikuttavat mm. lypsykauden vaihe ja utaretulehduksen aiheuttajabakteeri ja sen taudinaiheuttamiskyky. Laktoferriini tarjoaa potentiaalisia mahdollisuuksia utareen vastustuskyvyn lisäämiseen, mutta käyttökelpoisten sovellusten kehittäminen vaatii lisätutkimuksia. Siirtogeenisten lehmien maidon korkean laktoferriinipitoisuuden odotettua vähäisemmät positiiviset vaikutukset eivät rohkaise tämänkaltaisen geenimanipulaation käyttöön vastustuskykyisempien lypsylehmien jalostamisessa.