Samples of inoculum were taken, diluted in PBS, and spiral plated onto LB plates for colony remuneration. The first animal, a female, was mated and allowed to carry the pregnancy to term. 2017 Oct 10;12(10):e0186056. <> At least five villi were measured per slide. State of the art in the production of transgenic goats. The pigs fed hLZ-milk had a significantly thinner lamina propria layer (p = 0.0004) compared to control milk-fed pigs, indicating less inflammation (Table 2, Figure 4). No differences in ZO-1 relative expression patterns were observed in cultured cells, with increased expression in IL-6 in cells exposed to nTg milk than controls, with the Tg group being similar to all groups. A total of four doses were given, once every 12 hours. The hLZ-milk fed pigs also had deeper crypts, which contain the multicomponent local stem cells that give rise to terminally differentiated functional cells including enterocytes [38]. 224 0 obj When these transgenic goats have kids and start lactating, the researchers will collect the milk and purify the spider silk protein into “much, much higher quantities,” Lewis said. 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. 169 0 obj <> Epub 2017 Nov 8. 158 0 obj [1085 0 R 1086 0 R 1087 0 R 1088 0 R 1089 0 R 1090 0 R 1091 0 R 1092 0 R 1093 0 R 1094 0 R 1095 0 R 1096 0 R 1097 0 R 1098 0 R 1099 0 R 1100 0 R 1101 0 R 1102 0 R 1103 0 R 1104 0 R 1105 0 R 407 0 R 408 0 R 409 0 R 410 0 R 411 0 R 412 0 R 413 0 R 414 0 R 1106 0 R 1107 0 R 1108 0 R 1109 0 R 1110 0 R 1111 0 R] <> Pigs fed hLZ-milk had significantly lower proportion of circulating monocytes (p = 0.0413) at the end point of the study (Figure 3c). endobj At the end of the study, pigs fed control milk were significantly more dehydrated than animals fed hLZ-milk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https://doi.org/10.1371/journal.pone.0058409.g003. 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. Goat Genomic Resources: The Search for Genes Associated with Its Economic Traits. 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. <> 171 0 obj endobj Two lines were selected for detailed characterization. <> endobj <> endobj endobj endobj 2020-06-08T11:35:37+12:00 endobj IL-8 causes chemotaxis of leukocytes from peripheral lymphoid organs to the site of infection, and pigs fed hLZ-milk had significantly lower expression of IL-8 in the ileum than pigs fed control milk. endobj <> In both the duodenum and ileum, pigs fed hLZ-milk had significantly taller villi than those fed control milk (p = 0.0103, p<0.0001, respectively). 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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obj 205 0 obj 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). 154 0 obj 4 0 obj Yes Statistical analysis for fold expression differences from the qPCR assay was performed using REST-MCS software using β-actin as a housekeeping gene. endobj Department of Animal Science, University of California Davis, Davis, California, United States of America, Affiliations Is the Subject Area "Swine" applicable to this article? [356 0 R 357 0 R 358 0 R 359 0 R] endobj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endobj 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). Citation: Cooper CA, Garas Klobas LC, Maga EA, Murray JD (2013) Consuming Transgenic Goats' Milk Containing the Antimicrobial Protein Lysozyme Helps Resolve Diarrhea in Young Pigs. [252 0 R 253 0 R 253 0 R 253 0 R 253 0 R 253 0 R 253 0 R 253 0 R 253 0 R 254 0 R] 163 0 obj endobj 187 0 obj Here we report the characterization of the milk from 5 transgenic females of this line expressing human lysozyme in their milk at 270 microg/mL or 68% of the level found in human milk. [382 0 R 383 0 R 384 0 R 385 0 R 386 0 R 387 0 R 388 0 R] Histological sections at 10X magnification showing average villi from the ileum of pigs (A) fed hLZ-milk or (B) control milk. However, this is the first report that demonstrates that the hLZ-milk is an effective treatment for diarrhea caused by a bacterial infection in the gastrointestinal (GI) tract, fulfilling the promise of using transgenic animals to produce food with improved functional characteristics. Analyzed the data: CAC LCGK. <>stream All primers were porcine specific and validated before use (Table 1). endobj endobj For hemolytic bacteria control fed pigs had an average of 4.089±0.671 log (CFUs/gram)±S.D and hLZ-milk fed pigs had an average of 3.597±0.583 log (CFUs/gram)±S.D (p = 0.158). endobj <> 2016 Oct;25(5):575-95. doi: 10.1007/s11248-016-9965-1. 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. The potential for applying biotechnology to benefit animal agriculture and food production has long been speculated. No, Is the Subject Area "Diarrhea" applicable to this article? In 1999 the Artemis line of transgenic goats producing milk containing human lysozyme (hLZ-milk) was established [4]. J Dairy Sci. endobj 206 0 obj Find NCBI SARS-CoV-2 literature, sequence, and clinical content: https://www.ncbi.nlm.nih.gov/sars-cov-2/. endobj 189 0 obj Observation data was compared using the repeated measures function in SAS and the Greenhouse-Geisser Epsilon test to determine differences. Copyright: © 2013 Cooper et al. 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. 1991 Sep;9(9):839-43. doi: 10.1038/nbt0991-839. <> https://doi.org/10.1371/journal.pone.0058409.g002. endobj Front Microbiol. Pigs were genotyped for F4ab/ac which indicates early (<24 hours) or later (>24 hours) onset of E. coli infections and all pigs were homozygous for later onset [30]. Lysozyme is an important non-specific immune protein in human milk, modulating the immune response against bacterial infections. Pigs were euthanized 92 hours after the first dose of ETEC (Figure 1). Milk was pasteurized at 73.8°C then immediately cooled and stored at 4°C for no more than a … Biotechnology (N Y). 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. In the ileum, the control milk-fed pigs had short, wide villi, a sign that the villi had been damaged and were still in the process of repairing themselves. The addition of human milk components with intrinsic antimicrobial activity and positive charge to livestock milk by genetic engineering has the potential to benefit animal health, as well as food safety and production. Baldassarre H, Wang B, Keefer CL, Lazaris A, Karatzas CN. But as for the well-being of those goats, Dr Lewis says: “In over 15 years of breeding these goats, we have no indications of any abnormal health risks …We have simply transferred a gene for spider silk protein into the goat genome and have put it under the same cell regulation system as the other milk … 168 0 obj 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. [330 0 R 331 0 R 332 0 R 333 0 R 334 0 R 335 0 R 336 0 R 337 0 R 337 0 R 337 0 R 338 0 R 339 0 R 340 0 R 341 0 R 342 0 R]

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