In vitro growth of mastitis-inducing Escherichia coli in milk and milk fractions of dairy cows

Abstract

The outcome of E. coli mastitis in cows ranges from mild to severe in individual animals. This study explored the hypothesis that milk from individual cows differs in its growth medium properties for E. coli, and whether possible variation could be related to specific milk constituents. To mimic the early phase of intramammary E. coli infection, a low inoculum size and a short incubation period were used. Cell-reduced, cell- and fat-free (skim) and cell- and fat-free and protein-reduced (whey) fractions were prepared from whole milk samples (n=18). Ten ml of whole milk, milk fractions and brain heart infusion broth (BHI) were inoculated with approximately 100 cfu E. coli. After 6 h of incubation, bacterial counts were assessed by dilution plating in triplicate. Bacterial counts in whole milk differed up to a 100-fold between cows, which was not associated with SCC. Bacterial counts were significantly higher in whey fractions than in whole milk, cell-reduced and skim fractions and variation in whey was smaller, indicating that the acid-precipitable protein fraction contains the milk constituents of major relevance for inhibition of and variation in bacterial growth. The presence of fat and cells added to bacterial growth inhibition to a lesser extent. In conclusion, in vitro growth of E. coli in milk differs substantially between individual cows within an incubation period comparable with the early phase of intramammary infection. This suggests that the growth medium properties of milk could be of importance in the pathogenesis of E. coli mastitis and subsequent outcome of disease.

Introduction

Escherichia coli is an important pathogen causing clinical mastitis in dairy cows (Erskine et al., 1988, Barkema et al., 1998). The outcome of experimental E. coli mastitis ranges from mild to severe in individual dairy cows, and can be quantified by bacterial counts in milk after infusion (Lohuis et al., 1990). The number of bacteria in milk depends on the balance between the growth rate of bacteria and the elimination rate by host defense mechanisms.

Several authors have postulated that bacterial growth in milk is an important factor during the early stages of intramammary infection (Dutt et al., 1986, Fang et al., 1998; Leigh et al., 1990; Lammers et al., 2000). Differences in the severity of experimental E. coli mastitis, however, have mainly been attributed to individual variation in host defense mechanisms. Massive influx of polymorphonuclear leukocytes (PMN) from peripheral blood into milk and efficient killing of the bacteria by these PMN results in elimination of the pathogen after intramammary infusion (Burvenich et al., 1999). It takes as long as 7–9 h after experimental intramammary infusion of the bacteria before increased PMN numbers become apparent in milk (van Werven, 1999). This interval from infusion until PMN influx can be used by the bacteria to adapt to their environment and to multiply. Milk from individual cows could differ in its growth medium properties for E. coli, resulting in different levels of bacterial load within the first hours after experimental infusion, and ultimately in differences in severity of E. coli mastitis. An explanation for differences in bacterial growth in early infection could be the number of somatic cells present in milk (Shuster et al., 1996).

The aim of this study was to investigate whether in vitro growth of E. coli in milk differs between individual cows, and if so, whether this variation is associated with specific milk constituents. For this purpose, quantification of somatic cells, fat, protein and lactose in whole milk was performed, and milk fractions were prepared. The early phase of intramammary infection was mimicked by means of a low inoculum size and a short incubation period.