Consuming transgenic goats' milk containing the antimicrobial protein lysozyme helps resolve diarrhea in young pigs.

Childhood diarrhea is a significant problem in many developing countries and E. coli is a main causative agent of diarrhea in young children. Lysozyme is an antimicrobial protein highly expressed in human milk, but not ruminant milk, and is thought to help protect breastfeeding children against diarrheal diseases. We hypothesized that consumption of milk from transgenic goats which produce human lysozyme (hLZ-milk) in their milk would accelerate recovery from bacterial-induced diarrhea. Young pigs were used as a model for children and infected with enterotoxigenic E. coli. Once clinical signs of diarrhea developed, pigs were fed hLZ-milk or non-transgenic control goat milk three times a day for two days. Clinical observations and complete blood counts (CBC) were performed. Animals were euthanized and samples collected to assess differences in histology, cytokine expression and bacterial translocation into the mesenteric lymph node. Pigs consuming hLZ-milk recovered from clinical signs of infection faster than pigs consuming control milk, with significantly improved fecal consistency (p = 0.0190) and activity level (p = 0.0350). The CBC analysis showed circulating monocytes (p = 0.0413), neutrophils (p = 0.0219), and lymphocytes (p = 0.0222) returned faster to pre-infection proportions in hLZ-milk fed pigs, while control-fed pigs had significantly higher hematocrit (p = 0.027), indicating continuing dehydration. In the ileum, pigs fed hLZ-milk had significantly lower expression of pro-inflammatory cytokine IL-8 (p = 0.0271), longer intestinal villi (p<0.0001), deeper crypts (p = 0.0053), and a thinner lamina propria (p = 0.0004). These data demonstrate that consumption of hLZ-milk helped pigs recover from infection faster, making hLZ-milk an effective treatment of E. coli-induced diarrhea

Time line of experiment.

<p>Timeline includes dosing of ETEC, blood draws, and milk feeding schedule.</p

Pigs fed hLZ-milk are less dehydrated and clear the ETEC infection faster than control-fed pigs.

<p>Concentrations (mean ± SEM) of (<b>A</b>) hematocrit (<b>B</b>) hemoglobin (<b>C</b>) monocytes (<b>D</b>) neutrophils and (<b>E</b>) lymphocytes over the course of the study as determined by CBC analysis. Dunnett’s test was used for single comparisons between the hLZ-milk fed (n = 10) and control milk fed (n = 12) groups, *indicates <i>p</i><0.05. Pigs fed hLZ-milk suffer from less dehydration (significantly lower concentrations of hematocrit and hemoglobin) and return to baselines levels of circulating leukocytes (significantly fewer monocytes and lymphocytes, and significantly more neutrophils in circulation) compared to control-fed pigs.</p

Porcine specific primer sequences for qRT-PCR.

<p>Porcine specific primer sequences for qRT-PCR.</p

Expression levels of cytokines in the duodenum and ileum of hLZ-fed (<i>n</i> = 10) and control-fed animals (<i>n = 12)</i>.

*<p>Indicates significant differences between control-milk and hLZ-milk groups, <i>p</i><0.05.</p

Pigs fed hLZ- milk have improved fecal consistency and activity scores.

<p>Average scores (mean± SEM) for (A) fecal consistency and (B) activity level for the entire experiment. When analyzed from the start of milk treatments to the end of the study, pigs fed hLZ-milk (n = 10) had improved fecal consistency compared to pigs fed control milk (n = 12) (<i>p</i> = 0.0090), as well as an increased level of activity (<i>p</i> = 0.0350). Data was analyzed using the repeated measures function in SAS and the Greenhouse-Geisser Epsilon method to calculate <i>p</i> values *indicates <i>p</i><0.05.</p

Pigs fed hLZ-milk had less ETEC-induced intestinal damage at the end of the experiment.

<p>Histological sections at 10X magnification showing average villi from the ileum of pigs (<b>A</b>) fed hLZ-milk or (<b>B</b>) control milk. Pigs fed hLZ-milk had reduced damage due to ETEC infection as seen by increased surface area (taller villi) and less inflammation (thinner lamina propria), compared to pigs fed control milk.</p