Dissecting the early host response during pathogen-specific mastitis

The clinical picture of bovine mastitis is mostly related to the causative pathogen; however, the underlying reasons remain unclear. A deeper understanding of the pathogen-specific mechanisms leading to their divergent clinical pictures may be crucial in future for the development of new strategies for prophylaxis and therapy. One of the main research goals of Dr. Wolfram Petzl since 2003 has been to gain underpinning knowledge on the divergent pathophysiology of clinical and subclinical mastitis. Interdisciplinary research collaborations assembling the molecular basis of the early immune response during pathogen-specific mastitis formed the basis for a comprehensive view.

Dr. habil. Wolfram Petzl is senior researcher at the Clinic for Ruminants with Ambulatory and Herd Health Services at the Ludwig-Maxilians-Universität Munich, Germany

Bovine mastitis imposes major constraints on the dairy industry, affecting health and welfare, decreasing productivity and increasing production costs, as well as having an impact on public health through antibiotic usage and wastage. Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) represent two highly important mastitis pathogens related to subclinical and clinical mastitis respectively. Their divergent clinical pictures of disease make them promising candidates for comparative studies about the early immune response in the udder.

The immune response during mastitis is double-edged
Mastitis is the inflammatory response towards intramammary infection (IMI). A complex course of pathophysiological events determines whether the outcome results in clinical or subclinical mastitis. To what extent an adequate immune response should rise remains ambivalent and has to be considered carefully from two points of view. Although a pronounced inflammatory response during clinical mastitis may more likely resolve an IMI, it holds the risk of adversely affecting the cow’s well-being and

may result in a life-threatening toxic shock syndrome. In contrast, subclinical mastitis is often characterized by an inadequate weak immune response allowing pathogen persistence and long term production losses. From a simplified point of view, bovine mastitis becomes problematic when the initial immune response is either too weak or too strong.

Pathogen recognition is a decisive key event at the onset of mastitis
During IMI, bacterial components are recognised by pattern recognition receptors. This primary interaction of the innate arm of immune defences initiates a cascade of downstream events leading to the expression and release of antimicrobial molecules, chemokines and cytokines from host cells. All these factors contribute to an inflammatory response that is typical for either the causative mastitis pathogen or the present immune status of the cow. In consequence the outcome of the immune response is of key importance to the productiveness of the cow.

German mastitis research group promotes interdisciplinary dialogue on pathogen-specific mastitis
The key for a comprehensive view on the early immune response during pathogen-specific mastitis was the collaboration of different universities and research institutes. This conjoint approach made it possible to bring together experts from the fields of clinical veterinary medicine, immunology and molecular biology. The core group of the German mastitis consortium namely involves Wolfram Petzl and Holm Zerbe from the Clinic for Ruminants (Ludwig-Maximilians-University Munich), Hans-Joachim Schuberth (Immunology Unit, University of Veterinary Medicine, Hannover) and Juliane Günther and Hans-Martin Seyfert (Institute of Genome Biology, Leibniz Institute for Farm Animal Biology, Dummerstorf). Working within national and international research networks with other scientists, developing mastitis challenge models in vivo and in vitro and applying newly available holistic molecular methods enabled the group to gain a deeper understanding of responsible molecular mechanisms.

Mastitis models in vivo help elucidating pathogenesis
Understanding the complexity of the early host response during pathogen-specific mastitis requires suitable model systems. However, a significant amount of work could be achieved by in vitro assays using primary bovine mammary epithelial cells (pbMEC) the application of in vivo infection models is inevitable with regards to an early response in an entire host organism. Thus, the interdisciplinary holistic approaches demanded the development of stringent animal infection models. To minimize the cow-specific influence, a focus was laid on the selection and pre-screening of animals. To ensure comparable results and as little inter-individual variability as possible, only purebred Holstein-Frisian heifers in their third to fifth month of lactation were applied. All animals had a history free from mastitis and repeatedly tested negative for mastitis pathogens in their milk. Their somatic cell count (SCC) was <50.000cells/ml for at least three weeks prior to challenge. Animals were estrus synchronized and received the pathogen challenge with either E. coli or S. aureus in one udder quarter at day of estrus to rule out hormonal influences. The transcriptomic host response reflects the clinical picture of pathogen-specific mastitis The characteristic symptoms of E. coli- and S. aureus-specific mastitis could repeatedly be simulated in controlled in vivo challenge experiments without exception. All animals developed moderate clinical mastitis within 12 h after E. coli challenge. S. aureus challenge merely induced an increased SCC 12–60 hours after inoculation and a recurring shedding of S. aureus into the milk. The clinical picture of the disease was reflected by the sheer quantity of regulated genes in the infected udder tissue: animals that were culled 24 hours after E. coli challenge showed >2000 differentially expressed genes (DEG), whereas the transcriptomic response towards S. aureus challenge was virtually absent 24 hours after challenge and comparably weak (202 DEG) 72 hours after challenge. The vigorous transcriptional response during E. coli mastitis may explain the frequent spontaneous bacteriological cure, since the predominant part of regulated genes coded for innate inflammation related factors and endogenous antimicrobial factors. The lack of a comparable host response towards S. aureus, however, may contribute to persistence of S. aureus IMI.

The host response in the udder follows a spatiotemporal gradient
Mastitis research during the last decades has closely examined the pathophysiology of the inflamed mammary gland. However, little has been known about the first transcriptional response before the disease becomes apparent by clinical signs (e.g. fever, udder swelling) or the increase of SCC in milk. With regards to earliest transcriptional events a short-term infection model was applied where animals were culled 3–6 hours after pathogen challenge with E. coli or S. aureus. However, the results of the transcriptomic analysis were sobering, since there was no noteworthy response present in the udder tissue. By extending the locations for tissue sampling it became obvious that shortly after pathogen challenge the transcriptomic response was restricted to the very distal compartments of the udder (teat and milk cistern). Sequentially challenging three of four udder quarters within one animal allowing a pathogen presence of 1–3 hours before culling and tissue sampling made it possible to identify a set of genes which were regulated very early after S. aureus and E. coli challenge. A pathogen-specific response was characteristic already within the first hour after challenge. Again the response was dominated by factors of the innate immune response: transcripts for chemokines, cytokines and antimicrobial defence molecules showed to be the first to be regulated after pathogen contact. Furthermore, it clearly demonstrated that the course of the disease is already decided at the site of entry and the typical divergent host response patterns caused by E. coli and S. aureus with regards to clinical and subclinical mastitis are already reflected in quantity and quality of the transcriptomic response at this early stage in the teat.

Interdisciplinary collaboration isthe key to a comprehensive view on pathogen-specific mastitis

Dissecting local and systemic host response during clinical mastitis
Experimental mastitis models applying E. coli challenge revealed basically two distinct transcriptomic response patterns. One was restricted locally to the actually infected udder quarters (local response) whereas a second response could be carved out in infected and non-infected neighbouring udder quarters (systemic response). These different response patterns could only be identified, when comparing data of challenged and neighbouring non-challenged quarters of E. coli-challenged animals to untouched quarters of non-challenged animals. Non-challenged quarters of animals undergoing E. coli IMI in a neighbouring quarter showed a significant upregulation of e.g. acute phase proteins and antimicrobial factors. This observation suggested that the systemic host response during E. coli mastitis triggers the regulation of innate factors contributing to protecting the cow against secondary infections in the remaining udder quarters. Thus the systemic response during clinical mastitis may also change the udder quarter’s response to a following pathogenic threat. This was confirmed in a sequential animal infection model, where cows were consecutively challenged in two different udder quarters with a time lag of 12 hours. Whereas the first challenged udder quarter showed a full blown inflammatory response, no clinical mastitis could be induced in the secondary challenged quarter. This phenomenon is obviously based on a transient refractory state of the host, which results from repeated pathogen contact. This reflects the principle of endotoxin tolerance (ET) having a dual function. Besides an inhibition of inflammatory gene induction, endogenous antimicrobial factors undergo enhanced expression. This mechanism is regarded to protect the host towards new microbial infection and avoids overshooting inflammation during septicaemia.

Endotoxin tolerance as an immunomodulatory concept for mastitis prevention
During ET, mammary tissue undergoes a reprogramming, which very likely alters its pathogen-specific response. This transient effect is solely driven by factors of innate immunity and, like cases of natural IMI, does not induce a long-term protection against reinfection and recurrent mastitis cases. However, the equivocal effects of contemporary mastitis vaccination strategies aiming at long-term protection raise the question whether mechanisms of the adaptive immune actually response play a role in the mammary gland and can adequately be addressed. Immunomodulatory concepts could be aimed at ET to reduced harmful immune hyperreactivity during clinical mastitis, but keeping up a sustained antimicrobial environment. By inducing ET through treating the bovine mammary gland with very small amounts of Lipopolysaccharide (priming) and experimental E. coli challenge in vivo 3–10 days after priming this concept was proven. Animals were either protected against developing clinical mastitis (three days after priming) or the severity of mastitis was significantly being reduced (10 days after priming). Besides counteracting overshooting inflammation priming also led to a significantly enhanced bacterial clearance in milk of affected animals. Accompanying transcriptomic profiling of the priming concept in pbMEC in vitro revealed that mammary cells undergo ET by downregulation of proinflammatory factors while overexpressing genes for antimicrobial activity.

Outlook
This lately acquired knowledge about pathogen-specific local and systemic response and compartment-dependent early events during mastitis provide a basis for current research on immunomodulation and genetic markers targeting the innate immune system in the udder.
It still remains an open question as to how future immune modulatory concepts should address the dairy cow’s diverging immune phenotype at different stages of gestation and lactation. Epigenetics and its impact on udder health have recently gained more and more interest. Future research on epigenetic mechanisms during bovine mastitis may help facilitating the application of histone modifying enzymes for gene-specific control of inflammation.

Text and photos: Dr. Wolfram Petzl