Cytokine and acute phase protein gene expression in repeated liver biopsies of dairy cows with a lipopolysaccharide-induced mastitis

Abstract

A minimally invasive liver biopsy technique was tested for its applicability to study the hepatic acute phase response (APR) in dairy cows with Escherichia coli lipopolysaccharide (LPS)-induced mastitis. The hepatic mRNA expression profiles of the inflammatory cytokines, tumor necrosis factor α (TNF-α), IL-1β, IL-6, and IL-10, and the acute phase proteins serum amyloid A isoform 3 (SAA3), haptoglobin (Hp), and α1-acid glycoprotein (AGP) were determined by real-time reverse transcription-PCR. Fourteen primiparous cows in mid lactation were challenged with 200μg of LPS (n = 8) or NaCl solution (n = 6) in 1 front quarter. Six repeated liver biopsies were collected at −22, 3, 6, 9, 12, and 48h relative to LPS challenge in 4 LPS-infused cows and 3 NaCl-infused cows. The remaining cows had 3 liver biopsies taken at −22, 9, and 48h. Production data and clinical signs were recorded and white blood cell counts and somatic cell counts (SCC) were analyzed to investigate the effect of repeated liver biopsies and verify the LPS model. Plasma concentrations of TNF-α, SAA3, Hp, and AGP were determined for comparison with the liver expression data. Repeated liver biopsies had no effects on the production data, clinical signs, or APR of dairy cows. Compared with the NaCl-infused cows the LPS-infused cows responded to the LPS treatment by increased body temperature (38.6±0.1 vs. 39.4±0.1°C), short-term leukopenia followed by leukocytosis (6.44±0.4 vs. 5.69±0.3×106 cells/mL), an increased SCC (log10 2.1±0.1 vs. log10 2.8±0.1×103 cells/mL), heart rate (76±1 vs. 93±1 beats/min), and respiratory rate (32±2 vs. 36±1 breaths/min) in the acute phase of the disease. The LPS treatment upregulated the hepatic expression of TNF-α (103±24 vs. 255±18 units), IL-1β (37±23 vs. 296±18 units), IL-6 (8±17 vs. 122±12 units), and IL-10 (130±66 vs. 541±50 units), and SAA3 (64±36 vs. 128±28 units) and Hp (9±82 vs. 762±65 units) reaching maximum levels at 3 to 6h and 9 to 12h postinfusion, respectively. Plasma concentrations of TNF-α (nondetectable vs. 1.9±0.3 ng/mL), SAA (19.8±19.4 vs. 149.7±15.5μg/mL) and Hp (71.4±143.7 vs. 1,013.8±111.5μg/mL) were elevated in the LPS-infused cows at 4 to 12h, 8 to 120h, and 24 to 120h postinfusion, respectively. The hepatic expression of AGP and the AGP plasma concentration remained unaltered in LPS-induced cows. In conclusion, a minimally invasive liver biopsy technique can be used for studying the hepatic APR in diseased cattle. Lipopolysaccharide-induced mastitis resulted in a time-dependent production of inflammatory cytokines and SAA and Hp in the liver of dairy cows.