Recombinant soluble CD14 reduces severity of intramammary infection by Escherichia coli

Abstract

The interaction among gram-negative bacteria, the innate immune system, and soluble CD14 (sCD14) has not been well documented. The effect of recombinant bovine sCD14 (rbosCD14) on milk somatic cell count (SCC), bacterial clearance, and cytokine production was investigated by using a bovine intramammary Escherichia coli infection model. We first determined whether rbosCD14 would increase the SCC during a lipopolysaccharide (LPS) challenge. Three quarters of each of six healthy lactating cows were injected with either 0.3 microg of LPS, 0.3 microg of LPS plus 100 micro g of rbosCD14, or saline. In comparison with quarters injected with LPS alone, the SCC was twofold higher (P < 0.05) in quarters injected with LPS plus rbosCD14 after the challenge. We therefore hypothesized that when E. coli bacteria invade the mammary gland, sCD14 in milk would interact with LPS and rapidly recruit neutrophils from the blood to eliminate the bacteria before establishment of infection. To test this hypothesis, two quarters of each of nine healthy cows were challenged with either 50 CFU of E. coli plus saline or 50 CFU of E. coli plus 100 microg of rbosCD14. Quarters challenged with E. coli plus rbosCD14 had a more rapid recruitment of neutrophils, which was accompanied by a faster clearance of bacteria, lower concentrations of tumor necrosis factor alpha and interleukin-8 in milk, and milder clinical symptoms, than challenged quarters injected with saline. Results indicate that increasing the concentration of sCD14 in milk may be a potential strategy with which to prevent or reduce the severity of infection by coliform bacteria.

FIG. 1.

Milk somatic cell counts during an LPS-induced intramammary challenge. Three glands of each cow were challenged with either saline (▪), 0.3 μg of LPS plus saline (•), or 0.3 μg of LPS plus 100 μg of rbosCD14 (▴). Data are presented as the means ± the standard errors of the means of six cows. *, P < 0.05.

FIG. 2.

Appearance of mammary glands 24 h after an experimental challenge with 50 CFU of E. coli (A and B). The right glands exhibited more severe clinical symptoms, which included swelling, stiffness, and redness, than did the left glands, which also received 100 μg of rbosCD14. These pictures were randomly selected from among pictures of nine challenged animals. (C) Appearance of milk samples. The glands challenged with E. coli only produced yellowish and clumpy milk (right), in comparison with the normal-appearing milk produced by glands that received E. coli plus rbosCD14 (left).

FIG. 3.

Milk somatic cell response during experimentally induced intramammary E. coli infection. Two glands of each cow were challenged with 50 CFU of E. coli with (▴) or without (•) 100 μg of rbosCD14. The control glands received 100 μg of rbosCD14 in saline (▪). Data are presented as the means ± the standard errors of the means of nine cows. *, P < 0.05.

FIG. 4.

(A) Bacterial (CFU) counts in milk during an experimentally induced intramammary E. coli infection. Two glands of each cow were challenged with 50 CFU of E. coli with (▴) or without (•) 100 μg of rbosCD14. Data are presented as the means ± the standard errors of the means of nine cows. (B) Number of infected quarters after an E. coli challenge with (○) or without (•) 100 μg of rbosCD14.

FIG. 5.

Concentrations of TNF-α (A) and IL-8 (B) in milk during an experimentally induced intramammary E. coli infection. Two glands of each cow were challenged with 50 CFU of E. coli with (▴) or without (•) 100 μg of rbosCD14. Data are presented as the means ± the standard errors of the means of nine cows. *, P < 0.05; **, P < 0.01.