The authors have declared that no competing interests exist.
Conceived and designed the experiments: CAC EAM JDM. Performed the experiments: CAC LCGK. Analyzed the data: CAC LCGK. Contributed reagents/materials/analysis tools: CAC. Wrote the paper: CAC LCGK EAM JDM.
Childhood diarrhea is a significant problem in many developing countries and
Since the arrival of genetic engineering scientists have foreshadowed the use of transgenic animals for improving agriculture
Over a million children die each year from diarrheal diseases caused by bacterial and viral infections; with pathogenic strains of
The protein lysozyme is an important non-specific antimicrobial factor found in many bodily secretions. HEWL has been used for many years as a food additive and numerous studies have been conducted on the safety and effects of lysozyme. The FDA ruled HEWL as a generally recognized as safe (GRAS) compound in 1998
Lysozyme in human milk contributes to the establishment of stable bacterial populations within the gut, favoring the growth of beneficial bacteria such as
Many developing parts of the world rely on livestock as a main source of food, and through genetic engineering we can provide agriculturally relevant animals with novel traits targeted to help solve the problems facing these developing areas. As previously mentioned, we have generated a herd of transgenic goats that produce hLZ-milk
Milk was acquired from the UC-Davis goat facility. The hLZ-milk was collected and pooled from lactating does from the Artemis line of goats, while control goat milk was collected and pooled from non-transgenic control does. Milk was pasteurized at 73.8°C then immediately cooled and stored at 4°C for no more than a week before being fed to the pigs.
Six -week-old male Hampshire Yorkshire crossbred pigs, from a specific pathogen free facility were obtained. Pigs were genotyped for F4ab/ac which indicates early (<24 hours) or later (>24 hours) onset of
The O149:F4+ ETEC strain (Escherichia coli Laboratory, University of Montréal: 864075856716) was provided by Dr. John Fairbrother. The bacteria were cultured in a level 2 biosafety lab. One liter of TSB was inoculated and incubated at 37°C for 24 hours. Samples of inoculum were taken, diluted in PBS, and spiral plated onto LB plates for colony remuneration. The inoculum was then centrifuged at 4000 RPM for 10 minutes to isolate bacteria. The supernatant was removed and the bacteria were washed with PBS and spun again at 4000 RPM for 10 minutes. Supernatant was removed and the bacteria were suspended in saline solution. Pigs were orally dosed with bacteria suspended in saline solution.
A preliminary study was done to determine the dose of ETEC needed to cause diarrhea as well as monitor the progression of the clinical symptoms over time. Eight pigs were orally dosed with 2.5×10∧10 colony forming units (CFUs) of the porcine specific O149:F4+ ETEC. A total of four doses were given, once every 12 hours. This dosing regimen consistently caused sustained diarrhea in all pigs by 30 hours after the first dose, and by 72 hours after the first dose most pigs were starting to show signs of recovery. With this optimized dosing protocol two replicate milk treatment trials were conducted with 10 and 12 pigs respectively. Starting six hours after the final dose of ETEC was administered (42 hours), according to the groups they were previously assigned to, pigs were fed 250 mL of either pasteurized control goats’ milk or hLZ-milk three times daily for two days. Pigs were euthanized 92 hours after the first dose of ETEC (
Timeline includes dosing of ETEC, blood draws, and milk feeding schedule.
Blood was collected from the anterior vena cava into EDTA coated vacutainer tubes (Becton Dickinson Company, Franklin Lakes, NJ) before infection, 30 hours after the first dose of ETEC, and at the end of the study (92 hours after the first dose of ETEC). Complete blood count analyses on the samples were performed by Idexx Laboratories using an ADVIA 120 Hematology System (Siemens Healthcare Diagnostics Inc., Tarrytown, NY). A total of seventeen parameters were measured.
The fecal consistency and activity level of the pigs were assessed and recorded every twelve hours for the duration of the trial and feces were removed from the pens after each assessment. The same person, who was blinded to the treatments, conducted both observations. The scores were on a scale from zero to four. For fecal consistency a score of 4 indicated normal, solid feces, while a score of 0 was very watery feces. Similarly for activity scores a score of 4 was assigned to an alert and responsive pig that would react quickly to a person and to being fed while a score of 0 indicated a pig that was very lethargic and would refuse to move in response to being fed.
Pigs were euthanized using pentobarbital sodium (Fatal-Plus®, Vortech Pharmaceuticals, Ltd.) and tissue samples were collected. Duodenum samples were taken 20 cm below the pyloric sphincter and ileum samples were taken 20 cm above the ileocecal junction. Samples were also taken from the ileum mesenteric lymph node (MLN).
Samples from the duodenum and ileum were fixed in paraformaldehyde for 48 hours, then in 70% ethanol overnight. Samples underwent a process of step wise dehydration and were injected with paraffin (Tissue-Tek VIP 4 Tissue Processor, Sakura Int.). Paraffin injected samples were then embedded into paraffin to create blocks, which were subsequently cut into 6 µm sections and mounted onto slides. Slides were then stained with hematoxylin and eosin. Slides were analyzed by photographing then measuring the villi height, width, lamina propria thickness, and crypt depth at 10X magnification, using Spot Advanced Software (v3.4, Diagnostic Instruments). In addition, the number of lymphocytes and goblet cells per villus were counted at 40X magnification and analyzed as cells per unit villous height. At least five villi were measured per slide.
Total RNA was extracted from duodenum and ileum sections using Trizol reagent (Invitrogen, Carlsbad, CA) and was eluted in an appropriate amount of RNase-free water (Qiagen, Valencia, CA). For each sample, the integrity of the extracted RNA was analyzed by agarose gel electrophoresis by staining with ethidium bromide and visualization under UV light. Following extraction, RNA was treated with RQ1 DNase I (Promega, Madison WI) to remove any DNA contamination. Then the concentration was quantified and purity determined (OD260/OD280 absorption ratio >1.9) using a NanoDrop® ND-2000C Spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE). For each sample, 1 µg of RNA was converted to cDNA using OligoDT, 59 RT Buffer (Invitrogen), 0.1 M DTT,RNasin, dNTPs, DEPC dH20 and SuperScriptII reverse transcriptase (Invitrogen, Carlsbad, CA).
Quantitative real-time RT-PCR was performed using a 7500Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). Each reaction contained 12.5 µl of Fast SYBR Green (Applied Biosystems, Foster City, CA, USA) probe master mix, 1 µl of 10 µM reverses primer, 1 µl of 10 µM forward primer, 50 ng cDNA and dH20 in a total sample volume of 25 µl. Standard curves for each gene were constructed using cDNA template consisting of pooled cDNA from different pigs at concentrations of 300 ng/reaction with 1∶2 dilutions down to 4.68 ng/reaction. Amplification consisted of 3 steps: denaturation for 15 s at 94C°, annealing for 30 s at 60C°, and product extension for 30 s at 72C°. Fluorescence data was collected on the annealing step. This was repeated for a total of 40 cycles. Then product dissociation was measured in 3 steps: 95C° for 15 s, 60C° for 1 min, and 95C° for 15 s. For each plate, threshold and baseline were individually set and CT values determined using the 7500 Fast software. 40 cycles of qPCR at 95°C of denaturing for 15 s and 60°C of annealing/extension for 1 min. All reactions were conducted in triplicate. All primers were porcine specific and validated before use (
Gene name | Forward primer (5′-3′) | Reverse Primer (5′-3′) | Product (bp) | Acquisition number |
β-actin |
|
|
130 | U07786 |
IL-8 |
|
|
154 | M86923 |
IFN-γ |
|
|
146 | AY188090 |
TNF-α |
|
|
164 | X57321 |
IL-17 |
|
|
146 | NM_001005729 |
IL-10 |
|
|
150 | NM_214041 |
TGF-β |
|
|
164 | Y00111 |
Foxp3 |
|
|
149 | NM_001128438 |
Samples from the mesenteric lymph nodes were washed in sterile PBS. A 100 mg piece was weighed and homogenized. The homogenate was diluted in sterile PBS and plated on sheep blood agar plates. Plates were allowed to incubate at 37°C for 48 hours at which time the number of total and hemolytic colonies were counted.
Statistical evaluations for hematological, histological, observational, and bacterial data were conducted by use of the SAS statistical software, Version 9.3 (SAS Inc.,Cary, NC). Hematological, histological, and bacterial data were compared using the parametric test one-way analysis of variance (ANOVA) and differences were tested using the Dunnett's test. Observation data was compared using the repeated measures function in SAS and the Greenhouse-Geisser Epsilon test to determine differences. Statistical analysis for fold expression differences from the qPCR assay was performed using REST-MCS software using β-actin as a housekeeping gene. Differences were determined using the Pair Wise Fixed Reallocation Randomisation Test
Clinical signs of the illness became apparent in all pigs between 24 to 36 hours after the first dose of ETEC as the average fecal consistency and activity scores dropped, but once milk treatments started until the end of the study, pigs fed hLZ-containing milk had significantly higher fecal consistency (
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) (
At the end of the study, pigs fed control milk were significantly more dehydrated than animals fed hLZ-milk. Control-fed pigs had significantly higher hematocrit (
Concentrations (mean ± SEM) of (
In both the duodenum and ileum, pigs fed hLZ-milk had significantly taller villi than those fed control milk (
Histological sections at 10X magnification showing average villi from the ileum of pigs (
Duodenum | |||
Control milk | hLZ-milk | ||
|
429.92±150.07 | 493.67±143.34 |
|
|
150.00±38.90 | 164.79±55.75 | 0.2062 |
|
117.39±63.77 | 129.82±63.58 | 0.2825 |
|
476.63±173.78 | 434.06±145.87 | 0.1307 |
|
0.1615±0.0641 | 0.1402±0.0788 | 0.0990 |
|
0.0289±0.0236 | 0.0335±0.0177 | 0.4862 |
|
|||
|
|
|
|
|
431.76±136.41 | 560.10±112.72 |
|
|
166.48±128.49 | 135.91±41.14 | 0.0580 |
|
115.26±74.56 | 154.32±89.99 |
|
|
268.30±167.92 | 180.51±111.28 |
|
|
0.0997±0.0406 | 0.1078±0.0549 | 0.5956 |
|
0.0288±0.0134 | 0.0322±0.0138 | 0.2811 |
Measurements presented as mean ± S.D.
Indicates significant differences between control-milk and hLZ-milk groups,
At 92 hours after the first dose of ETEC, expression of the pro-inflammatory cytokine IL-8 was significantly lower in the ileum of pigs fed hLZ-milk compared to those fed control milk (
Gene name | Area of the gut | B-actin adjusted CT value | Fold change in expression | ||
Control milk | hLZ-milk | ||||
IL-8 | Duodenum | 32.74 | 32.94 | 0.226 | NA |
Ileum | 32.51 | 33.66 | 0.027 |
|
|
IFN-γ | Duodenum | 32.33 | 32.38 | 0.670 | NA |
Ileum | 32.46 | 32.32 | 0.256 | NA | |
TNF-α | Duodenum | 32.55 | 32.65 | 0.269 | NA |
Ileum | 32.38 | 32.60 | 0.715 | NA | |
IL-17 | Duodenum | 33.28 | 33.73 | 0.606 | NA |
Ileum | 33.32 | 33.76 | 0.617 | NA | |
IL-10 | Duodenum | 32.46 | 32.87 | 0.541 | NA |
Ileum | 33.61 | 34.02 | 0.705 | NA | |
TGF-β | Duodenum | 35.60 | 36.01 | 0.839 | NA |
Ileum | 35.59 | 36.09 | 0.812 | NA | |
Foxp3 | Duodenum | 29.55 | 29.47 | 0.430 | NA |
Ileum | 28.33 | 28.51 | 0.834 | NA |
Indicates significant differences between control-milk and hLZ-milk groups,
To assess the integrity of the intestinal epithelial cell tight junctions and quantify the amount of bacteria that migrated into the MLN, a bacterial translocation assay was conducted
Based on the results of these assays, we conclude that hLZ-milk is an effective treatment for a diarrhea caused by ETEC infection. This is supported by clinical observations, hematological, histological, and transcriptional data demonstrating that the ETEC infection was resolved more rapidly in pigs fed hLZ-milk in comparison to pigs fed control milk. Specifically, clinical observations showed that pigs fed with control milk continued to have watery feces, which was confirmed by the CBC analysis showing that control milk fed pigs had higher concentrations of hematocrit and hemoglobin, which are both indicative of dehydration. Clinical observations showed that control-fed pigs were also less active, another common symptom of both an acute phase response to infection and to dehydration.
Diarrhea is caused by the influx of water and ions into the intestinal lumen, and this persistent water loss causes dehydration. This influx is caused by disruption of ion transporters
The CBC analysis revealed that feeding hLZ-milk significantly decreased the demand for leukocytes, specifically monocytes, lymphocytes, and neutrophils at the site of infection. Splenic monocytes are recruited into circulation during an infection and home to the foci of inflammation. Thus, monocytemia is indicative of the acute phase of bacterial infection. Neutrophils are also quickly recruited to the site of a bacterial infection through chemotaxis, in response to pro-inflammatory cytokines such as IL-8 and INF-γ. Pigs do not store neutrophils thus there is a small pool of circulating neutrophils that are recruited out of circulation during an infection that can only be replaced by newly produced neutrophils from hematopoietic cells in the bone marrow. As an infection is cleared, the demand for neutrophils decreases, thus less are recruited from circulation, allowing circulating proportions to return to normal, however if an infection persists neutrophils will continue to decrease.
Lymphocytes are a key cell type involved in directing and propagating the adaptive immune response. After maturation they reside in the lymph nodes, spleen, and other lymphatic tissue. Circulating lymphocytes increased over the course of the infection and continued to rise in pigs fed control milk, while in pigs fed hLZ-milk the proportion of circulating lymphocytes returned closer to baseline amounts, indicating that pigs fed hLZ-milk had less demand for lymphocytes.
The pro-inflammatory cytokine IL-8 attracts macrophages, neutrophils, polymorphonuclear leukocytes (PMNs), and phagocytes as a first step in recruitment and induction of an inflammatory response
During an inflammatory reaction in the intestine the lamina propria is especially responsive to inflammation. In the ileum, the lamina propria of control fed pigs was significantly thicker, indicating continued inflammation
Overall, pigs that were fed hLZ-milk as a treatment for an intestinal ETEC infection recovered faster than pigs fed control milk. They were better hydrated, had less intestinal inflammation, more rapidly returned to normal proportions of blood leukocytes, and suffered less damage to their intestinal villi. This evidence demonstrates that feeding hLZ-milk at the onset of an intestinal ETEC infection is an effective treatment. Since lysozyme is part of the innate immune system, humans are constantly exposed to it through endogenous secretions including saliva and intestinal mucus, making it an ideal supplementary antimicrobial protein because it poses no risk as an allergen or toxin. For example, breast milk contains high concentrations of lysozyme and breastfeeding is a recommended treatment for diarrhea, however when breastfeeding is not possible oral rehydration therapy is often used. Oral rehydration therapy only addresses some of the symptoms of diarrhea, specifically loss of fluids and electrolytes. Ruminant milk containing hLZ helps the body remedy the infection faster than ruminant milk alone, restoring both the absorptive and barrier functions of the intestine, while providing fluids, electrolytes, and nutrients, making it a more comprehensive treatment for diarrhea than oral rehydration therapy. Based on these results, hLZ-milk from transgenic goats of the Artemis line could provide a safe, sustainable, and direct source of lysozyme-rich milk for communities facing high rates of childhood diarrhea.
We would like to thank Dr. John Fairbrother for providing the strain of ETEC, and Dr. James S. Cullor and Lynn Perani for assistance culturing the bacteria. We would also like to thank Jan Carlson and the UC-Davis Goat Barn staff for care and milking of dairy goats and Kent Parker and the UC-Davis Swine facility staff for assistance with pig rearing as well as Steve Vito, Leslie Stewart, and Elizabeth McInnis for help handling the pigs. We thank David Welch, Katherine Cubbon, and Brigette Santamaria for help examining the histological slides and Samantha Lotti for help processing intestinal samples for qRT-PCR.