Effects of recombinant bovine interleukin-8 (rbIL-8) treatment on health, metabolism, and lactation performance in Holstein cattle I: Production and functional characterization of rbIL-8 in vitro and in vivo

Abstract

In the present study, we standardized processes of cloning and purification of recombinant bovine interleukin-8 (rbIL-8) from bacterial culture and assessed its biological activity in Holstein cattle. Plasmid containing a subclone of bovine IL-8 was expressed using Escherichia coli BL21 and cell lysate was purified by chromatography. The presence of rbIL-8 was assessed by Western blot analyses and function was confirmed in vitro using a chemotaxis chamber. Based on optical density values, chemoattractant properties of rbIL-8 were 10-fold greater compared with control wells. Two in vivo studies were conducted to assess the biological activity of rbIL8. For study 1, one-year-old Holstein heifers (n = 20) were randomly allocated to receive a single intravaginal administration containing 1,125 µg of rbIL-8 diluted in 20 mL of saline solution (rbIL-8, n = 10) or a single intravaginal administration of 20 mL of saline solution (control, n = 10). For study 2, nonpregnant lactating Holstein cows (n = 31) were randomly allocated to receive an intrauterine administration with 1,125 µg of rbIL-8 diluted in 20 mL of saline solution (rbIL-8, n = 11), a positive control consisting of resin-purified lysate of E. coli BL21 not transfected with the plasmid coding for rbIL-8 diluted in 20 mL of saline solution (E. coli, n = 10), and a negative control administered with 20 mL of saline solution (control, n = 10). An increase in vaginal neutrophils was observed in heifers treated with rbIL-8 within 3 h of treatment, but not in control heifers. Additionally, intrauterine administration of rbIL-8 increased the proportion of PMN cells in uterine cytological samples from 3.5% before treatment to 75.8% 24 h later-an increase that was not observed in the negative control group and cows treated with resin-purified lysate of E. coli. To further evaluate the effect of local and systemic rbIL-8 stimulation on the dynamics of circulating white blood cells, a third study was conducted. In study 3, nonpregnant 8-mo-old Holstein heifers (n = 30) were randomly allocated into 1 of 3 treatment groups: intravenous rbIL-8 (1,125 µg of rbIL-8 diluted in 5 mL of saline solution, n = 10); intravaginal rbIL-8 (1,125 µg of rbIL-8 diluted in 20 mL of saline solution; n = 10); or intravaginal saline (20 mL of saline solution, n = 10). Intravenous injection of rbIL-8 resulted in a transient increase in rectal temperature, which was greater at 2 h after treatment compared with cows treated intravaginally with rbIL-8 or heifers treated with saline solution. Heifers treated with rbIL-8 intravenously displayed a marked reduction in neutrophils, basophils, lymphocytes, and monocytes within the first 4 h posttreatment compared with heifers treated intravaginally. However, at 6 h after treatment, heifers treated with rbIL-8 intravenously displayed a rebound in white blood cell counts caused by an increase in neutrophil counts. These results show that the presented purification method is effective and results in biologically active rbIL-8 that can be used safely to m...

Effects of recombinant bovine interleukin-8 (rbIL-8) treatment on health, metabolism, and lactation performance in Holstein cattle II: Postpartum uterine health, ketosis, and milk production

“…We demonstrated that our rbIL-8 molecule elicited the expected biological responses: strong chemoattractant properties in vitro and in vivo (following intravaginal and intrauterine administrations). More importantly, systemic, intrauterine, and intravaginal administration of rbIL-8 did not result in detectable undesirable side effects such as fever, increased respiratory rate, dehydration, or signs of pain and discomfort (Bicalho et al, 2019).…”

“…Our group has developed a recombinant bovine interleukin-8 (rbIL-8) molecule with the original objective of improving postpartum uterine health (Bicalho et al, 2019). We conducted a series of experiments to evaluate the safety of rbIL-8 administration and to assess its biological activity in vitro and in vivo.…”

Reproductive Tract Infections in Dairy Cows: Can Probiotics Curb Down the Incidence Rate?

“…Interleukin-8 is known for his chemoattractant properties toward neutrophils. Intrauterine infusion of recombinant bovine IL-8 (rbIL-8) by [143,144] within 24 h postpartum was reported to increase proportion of neutrophils in the vagina and uterine lumen and lower the prevalence of puerperal metritis in multiparous cows but had no effect on the incidence of retained placenta. The reader is referred to [145] for more details on this field of research.…”

“…It should be noted that the microbiota of the vaginal tract of dairy cows is not dominated by Lactobacillus strains as is the vaginal tract of female subjects [148]. There is also a difference in the vaginal pH between dairy cows and human vaginal pH, with cows having a slightly acidic to slightly alkaline neutral or slightly alkaline value (pH 6.8-7.35) and humans having an acidotic vaginal pH (3.5-4.5) [143]. Lactobacilli are known to produce lactic acid through hydrolysis of glycogen present in vaginal mucosa and lower the vaginal pH to acidic values, which is unique to humans [148].…”

“…Lactobacilli are known to produce lactic acid through hydrolysis of glycogen present in vaginal mucosa and lower the vaginal pH to acidic values, which is unique to humans [148]. Acidic vaginal pH plays a significant role in the prevention of bacterial infection [143]. It should be noted that, Lactobacilli use glycogen, released by vaginal epithelial cells to produce lactic acid.…”