Therapeutic and Prophylactic Effect of the Experimental Bacteriophage Treatment to Control Diarrhea Caused by E. coli in Newborn Calves

Abstract

The prevalence of antibiotic-resistant bacteria causing neonatal diarrhea in calves has become a serious problem in the control of infection. Due to increasing antibiotic resistance, bacteriophages with probiotics are considered the best alternative. The aim of the study was to evaluate the use of a suppository containing probiotic strains of Lactobacillus spp. and bacteriophages specific for pathogenic E. coli in young calves with diarrhea. The study evaluated therapeutic and prophylactic effects (specific and nonspecific humoral response). The study was carried out on 24 female HF calves, aged 2 to 7 days and weighing from 35 to 46 kg. The calves were divided into four groups (n = 6) as follows: Group 1, healthy control that received no medicine; Group 2, positive control with diarrhea; Group 3, healthy calves that received medicine; Group 4, calves with diarrhea that received medicine. The animals received suppositories containing Lactobacillus spp. and bacteriophages specific for pathogenic E. coli for 5 days. On the first day, the calves received the suppositories twice-in the morning and 12 h later; subsequently they were administered once a day. The health status of the calves was observed for 11 days after the first application of suppositories. A protective and preventive effect of the experimental therapy was obtained in the research. The probiotic-phage suppositories reduced the duration of diarrhea in calves, completely eliminating it within 24-48 h after use. The therapy stimulated the activation of immune mechanisms in calves, which translated into an enhanced specific and nonspecific response and increased resistance to infection.

Keywords: bacteria; bacteriophages; calves; diarrhea; therapy.

Figure 1

Detection of virulence genes in E. coli isolates used for in vitro and in vivo testing by multiplex PCR. Legend: M, molecular weight marker (100–1000 bp); E. coli strain numbers are given in individual lines.

Figure 2

Pulsed-field electrophoresis (PFGE) of undigested phage DNA. Legend: The lanes contained: 1, Marker II (485–48.4 kb), 2, φ26; 3, φ27; 4, φ29.

Figure 3

Dot plot analysis of average serum IgG and IgA concentrations in all experimental groups of calves. Legend A: a, significant difference at p ≤ 0.05 in comparison to untreated calves p = 0.04. Legend B: a, significant difference in comparison to untreated calves [p = 0.04]; post hoc effect analysis of variance, Levene’s test, significant differences at p = 0.029 in comparison to untreated calves; b, significant differences between heathy treated and diarrheic calves p = 0.048; c, significant differences between healthy treated and diarrheic treated calves p = 0.008; d, significant differences at p ≤ 0.05 between healthy untreated and diarrheic untreated calves p = 0.008

Figure 4

Correlation between IgG concentration, health status, and administration of medicine.

Figure 5

Correlation between IgA concentration, health status, and administration of medicine

Figure 6

Dot plot analysis of average serum concentrations of lysozyme and INFγ in experimental groups of calves. Legend A: a, significant difference between healthy treated and healthy untreated calves p = 0.00001; b, significant differences between healthy untreated and diarrheic untreated calves p = 0.0001; c, significant differences between healthy treated vs diarrheic treated calves [p = 0.006]; d, significant differences between diarrheic treated calves vs untreated calves [p = 0.008]. Legend B: a, significant differences at p ≤ 0.05 between treated and untreated calves (p = 0.0018); b, significant differences at p ≤ 0.05 between healthy calves and calves with diarrhea (p = 0.008); c, significant differences at p ≤ 0.05 between treated healthy and treated diarrheic calves.

Figure 7

Dot plot analysis of average plasma SAA and haptoglobin concentration in experimental groups of calves. Legend A: a, significant differences at p ≤ 0.05 for healthy treated vs healthy untreated calves; b, significant differences for treated calves with diarrhea vs untreated calves with diarrhea; c, significant differences for healthy untreated calves vs diarrheic treated calves; d, significant differences at p ≤ 0.05 for healthy treated calves vs diarrheic treated calves. Legend B: a, significant differences in analysis of variance at p ≤ 0.05 for diarrheic treated vs diarrheic untreated calves p = 0.0054; b, significant differences for healthy treated vs healthy untreated calves p = 0.0013; c, significant differences for healthy treated vs diarrheic calves p = 0.005; d, significant differences at p ≤ 0.05 for healthy treated vs diarrheic untreated calves.