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1.  Changes in the gut microbiota of cloned and non-cloned control pigs during development of obesity: gut microbiota during development of obesity in cloned pigs 
BMC Microbiology  2013;13:30.
Background
Obesity induced by a high-caloric diet has previously been associated with changes in the gut microbiota in mice and in humans. In this study, pigs were cloned to minimize genetic and biological variation among the animals with the aim of developing a controlled metabolomic model suitable for a diet-intervention study. Cloning of pigs may be an attractive way to reduce genetic influences when investigating the effect of diet and obesity on different physiological sites. The aim of this study was to assess and compare the changes in the composition of the gut microbiota of cloned vs. non-cloned pigs during development of obesity by a high-fat/high-caloric diet. Furthermore, we investigated the association between diet-induced obesity and the relative abundance of the phyla Firmicutes and Bacteroidetes in the fecal-microbiota. The fecal microbiota from obese cloned (n = 5) and non-cloned control pigs (n= 6) was investigated biweekly over a period of 136 days, by terminal restriction fragment length polymorphism (T-RFLP) and quantitative real time PCR (qPCR).
Results
A positive correlation was observed between body-weight at endpoint and percent body-fat in cloned (r=0.9, P<0.0001) and in non-cloned control pigs (r=0.9, P<0.0001). Shannon Weaver and principal component analysis (PCA) of the terminal restriction fragments (T-RFs) revealed no differences in the bacterial composition or variability of the fecal microbiota between the cloned pigs or between cloned and non-cloned control pigs. Body-weight correlated positively with the relative abundance of Firmicutes in both cloned (r=0.37; P<0.02) and non cloned-control pigs (r=0.45; P<0.006), and negatively with the abundance of Bacteroidetes in cloned pigs (r=−0.33, P<0.04), but not in the non-cloned control pigs.
Conclusion
The cloned pigs did not have reduced inter-individual variation as compared to non-cloned pigs in regard to their gut microbiota in neither the obese nor the lean state. Diet-induced obesity was associated with an increase in the relative abundance of Firmicutes over time. Our results suggest that cloned pigs are not a more suitable animal model for gut microbiota-obesity related studies than non-cloned pigs. This study is the first to evaluate if cloned pigs provide a better animal model than conventional pigs in diet-intervention, obesity and gut microbiota research.
doi:10.1186/1471-2180-13-30
PMCID: PMC3610253  PMID: 23391125
Gut microbiota; Cloned pigs; Diet-induced obesity; Bacterial diversity; Bacteroidetes; Firmicutes
2.  Cloning Changes the Response to Obesity of Innate Immune Factors in Blood, Liver, and Adipose Tissues in Domestic Pigs 
Cellular Reprogramming  2013;15(3):185-194.
Abstract
The objective of this study was to evaluate the usefulness of cloned pigs as porcine obesity models reflecting obesity-associated changes in innate immune factor gene expression profiles. Liver and adipose tissue expression of 43 innate immune genes as well as serum concentrations of six immune factors were analyzed in lean and diet-induced obese cloned domestic pigs and compared to normal domestic pigs (obese and lean). The number of genes affected by obesity was lower in cloned animals than in control animals. All genes affected by obesity in adipose tissues of clones were downregulated; both upregulation and downregulation were observed in the controls. Cloning resulted in a less differentiated adipose tissue expression pattern. Finally, the serum concentrations of two acute-phase proteins (APPs), haptoglobin (HP) and orosomucoid (ORM), were increased in obese clones as compared to obese controls as well as lean clones and controls. Generally, the variation in phenotype between individual pigs was not reduced in cloned siblings as compared to normal siblings. Therefore, we conclude that cloning limits both the number of genes responding to obesity as well as the degree of tissue-differentiated gene expression, concomitantly with an increase in APP serum concentrations only seen in cloned, obese pigs. This may suggest that the APP response seen in obese, cloned pigs is a consequence of the characteristic skewed gene response to obesity in cloned pigs, as described in this work. This should be taken into consideration when using cloned animals as models for innate responses to obesity.
doi:10.1089/cell.2012.0091
PMCID: PMC3666226  PMID: 23668862
3.  The effect of high-fat diet on the composition of the gut microbiota in cloned and non-cloned pigs of lean and obese phenotype 
Gut Microbes  2013;4(5):371-381.
The aim of this study was to investigate the effect of high-far-high-energy diet on cloned and non-cloned domestic pigs of both lean and obese phenotype and to evaluate if the lean cloned pigs had a lower inter-individual variation as compared with non-cloned pigs. The microbiota of colon and terminal ileum was investigated in cloned and non-cloned pigs that received a high-far-high-energy diet with either restricted or ad libitum access to feed, resulting in lean and obese phenotypes, respectively. The fecal microbiota of lean pigs was investigated by terminal restriction fragment length polymorphism (T-RFLP). The intestinal microbiota of lean and obese cloned and non-cloned pigs was analyzed by quantitative real time PCR and a novel high-throughput qPCR platform (Fluidigm). Principal component analysis (PCA) of the T-RFLP profiles revealed that lean cloned and non-cloned pigs had a different overall composition of their gut microbiota. The colon of lean cloned pigs contained relatively more bacteria belonging to the phylum Firmicutes and less from the phylum Bacteroidetes than obese cloned pigs as estimated by qPCR. Fluidigm qPCR results revealed differences in specific bacterial groups in the gut microbiota of both lean and obese pigs. Our results suggest that high-far-high-energy diet is associated with changes in the gut microbiota even in the absence of obesity. Overall, the cloned pigs had a different gut microbiota from that of non-cloned pigs. To our knowledge this is the first study to investigate the gut microbiota of cloned domestic pigs of lean and obese phenotype.
doi:10.4161/gmic.26108
PMCID: PMC3839981  PMID: 23974297
cloned pigs; gut microbiota; high-fat–high-energy diet; inter-individual variation; obesity
4.  Embryonic Pig Pancreatic Tissue Transplantation for the Treatment of Diabetes 
PLoS Medicine  2006;3(7):e215.
Background
Transplantation of embryonic pig pancreatic tissue as a source of insulin has been suggested for the cure of diabetes. However, previous limited clinical trials failed in their attempts to treat diabetic patients by transplantation of advanced gestational age porcine embryonic pancreas. In the present study we examined growth potential, functionality, and immunogenicity of pig embryonic pancreatic tissue harvested at different gestational ages.
Methods and Findings
Implantation of embryonic pig pancreatic tissues of different gestational ages in SCID mice reveals that embryonic day 42 (E42) pig pancreas can enable a massive growth of pig islets for prolonged periods and restore normoglycemia in diabetic mice. Furthermore, both direct and indirect T cell rejection responses to the xenogeneic tissue demonstrated that E42 tissue, in comparison to E56 or later embryonic tissues, exhibits markedly reduced immunogenicity. Finally, fully immunocompetent diabetic mice grafted with the E42 pig pancreatic tissue and treated with an immunosuppression protocol comprising CTLA4-Ig and anti–CD40 ligand (anti-CD40L) attained normal blood glucose levels, eliminating the need for insulin.
Conclusions
These results emphasize the importance of selecting embryonic tissue of the correct gestational age for optimal growth and function and for reduced immunogenicity, and provide a proof of principle for the therapeutic potential of E42 embryonic pig pancreatic tissue transplantation in diabetes.
Editors' Summary
Background.
Diabetes is a growing global health problem. By 2030, more than 300 million people around the world will have this chronic, incurable disorder, double the current number. In non-diabetic people, cells in the pancreas called beta cells release insulin, a hormone that controls the level of sugar (glucose) in the blood. In diabetics, blood-sugar levels become dangerously high either because the beta cells have been destroyed so no insulin is made (type 1 diabetes, 5%–10% of all cases) or because the cells that normally remove sugar from the blood have become insensitive to insulin (type 2 diabetes). In particularly severe cases of type 2 diabetes, the beta cells also stop releasing insulin. People with type 2 diabetes can usually control their blood-sugar levels through diet and exercise and by taking oral anti-diabetic drugs; people with type 1 diabetes or severe type 2 diabetes have to replace the missing insulin by injections. It is very important that diabetics keep their blood-sugar levels as normal as possible to minimize the disorder's serious long-term complications. These include kidney failure, blindness, nerve damage, and an increased risk of heart disease and strokes.
Why Was This Study Done?
While individuals with type 1 diabetes can control their blood-sugar levels pretty well by carefully monitoring their life style and injecting insulin, potentially better control and fewer long-term complications can be achieved by providing a new source of insulin-producing cells through transplantation of pancreatic tissue from a dead human donor. However, because there is not enough human pancreatic tissue to treat all the diabetics who could benefit from such transplants, researchers are investigating other sources of insulin-producing cells. One possibility is pig pancreatic tissue. Glucose control is very similar in pigs and humans, pig insulin injections have been used for years to control diabetes, and pigs are in plentiful supply. However, besides general concerns about xenotransplantation (that is, transplantation from a foreign species such as pigs into humans), early attempts to treat human diabetes by transplantation of pancreatic tissue taken from pig embryos at late stages of gestation were not successful. The researchers involved in this study had done earlier experiments that suggested that the age of the pig donor tissue influences how well transplantation into other species works. They therefore wanted to test whether pancreatic tissue from younger pig embryos might work better for pancreas transplants: they hoped that younger tissue would grow and integrate better with the surrounding host tissue. Additionally, a major concern with all transplantations is whether the transplanted cells or tissue will be recognized as foreign and as such destroyed by the host's immune system. Because tissue from younger embryos is generally less likely to trigger an immune reaction, the researchers hoped that pancreatic tissue from younger pig embryos would be less readily recognized as foreign by the human immune system.
What Did the Researchers Do and Find?
They started by transplanting pancreatic tissue from pig embryos of different ages into mice with defective immune systems. Tissue taken about a third of the way through gestation (that is, from embryos 42 or 56 days old) grew better than tissue taken earlier or later, secreted more pig insulin over extended periods of time, and was better at maintaining normal blood-sugar levels when the beta cells of the host mice were destroyed. The researchers then examined whether embryonic pig pancreatic tissue of different ages triggered an immune reaction by seeing how well it survived when human immune system cells were also transplanted into the mice. Tissue from 42-day-old embryos came out best in this test too, suggesting that there is little or no “direct” immune reaction by circulating immune cells against pancreatic tissue from this stage. Finally, the researchers transplanted pancreatic tissue of this age into diabetic mice with an intact immune system. These mice rejected the transplants (presumably through an “indirect” immune reaction), but that rejection could be overcome when the recipient mice were treated with drugs that suppressed the part of their immune system that is responsible for these indirect immune reactions. (Human patients who receive a transplant are usually treated with drugs that suppress direct and indirect immune reactions.) When the mice were kept on the drugs, the grafts survived in the long term, and the mice had normal blood-sugar levels once the graft was well established.
What Do These Findings Mean?
These results suggest that the exact age of embryonic pig pancreatic tissue influences how well the transplanted tissue grows and integrates into a host from a different species (in this case, the mouse) and how strong an immune reaction it triggers. Overall, these results support the notion that pig embryonic pancreas tissue could potentially be a source of tissue for transplantation into human patients with diabetes. The next steps in exploring this possibility are likely to involve experiments in monkeys to find out how much tissue should be implanted and where, and to check that the transplanted tissue remains functional in these animals. The ability of the 42-day-old embryonic tissue to avoid direct immune rejection also needs to be confirmed. And, ideally, the goal remains to find ways to avoid an immune reaction altogether, so that recipients of transplants do not need to be continually treated with drugs that suppress their immune system (which makes them more susceptible to infections and can have other side effects). Xenotransplantation has potential benefits and risks and remains controversial. Studies like this one and others that seek to better understand the risks and benefits are necessary to allow reasonable decisions to be made.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030215
• MedlinePlus pages on diabetes and on pancreas transplantation
• Information from the Juvenile Diabetes Research Foundation International Description
• Wikipedia pages on diabetes, xenotransplantation, and pancreas transplantation (note: Wikipedia is a free online encyclopedia that anyone can edit)
Pancreatic tissue from embryonic pigs co-transplanted with or without human immune cells into immune-deficient mice suggests that the embryonic stage of the pig donor affects the immunogenicity of the graft.
doi:10.1371/journal.pmed.0030215
PMCID: PMC1479387  PMID: 16768546
5.  Comparison of Gene Expression and Genome-Wide DNA Methylation Profiling between Phenotypically Normal Cloned Pigs and Conventionally Bred Controls 
PLoS ONE  2011;6(10):e25901.
Animal breeding via Somatic Cell Nuclear Transfer (SCNT) has enormous potential in agriculture and biomedicine. However, concerns about whether SCNT animals are as healthy or epigenetically normal as conventionally bred ones are raised as the efficiency of cloning by SCNT is much lower than natural breeding or In-vitro fertilization (IVF). Thus, we have conducted a genome-wide gene expression and DNA methylation profiling between phenotypically normal cloned pigs and control pigs in two tissues (muscle and liver), using Affymetrix Porcine expression array as well as modified methylation-specific digital karyotyping (MMSDK) and Solexa sequencing technology. Typical tissue-specific differences with respect to both gene expression and DNA methylation were observed in muscle and liver from cloned as well as control pigs. Gene expression profiles were highly similar between cloned pigs and controls, though a small set of genes showed altered expression. Cloned pigs presented a more different pattern of DNA methylation in unique sequences in both tissues. Especially a small set of genomic sites had different DNA methylation status with a trend towards slightly increased methylation levels in cloned pigs. Molecular network analysis of the genes that contained such differential methylation loci revealed a significant network related to tissue development. In conclusion, our study showed that phenotypically normal cloned pigs were highly similar with normal breeding pigs in their gene expression, but moderate alteration in DNA methylation aspects still exists, especially in certain unique genomic regions.
doi:10.1371/journal.pone.0025901
PMCID: PMC3191147  PMID: 22022462
6.  Hierarchical Phenotypic and Epigenetic Variation in Cloned Swine1 
Biology of reproduction  2003;69(2):430-436.
Cloning by somatic cell nuclear transfer can result in the birth of animals with phenotypic and gene expression abnormalities. We compared adult cloned pigs and adult pigs from naturally bred control females using a series of physiological and genetic parameters, including detailed methylation profiles of selected genomic regions. Phenotypic and genetic analyses indicated that there are two classes of traits, one in which the cloned pigs have less variation than controls and another characterized by variation that is equally high in cloned and control pigs. Although cloning creates animals within the normal phenotypic range, it increases the variability associated with some traits. This finding is contrary to the expectation that cloning can be used to reduce the size of groups involved in animal experimentation and to reproduce an animal, including a pet, with a homogenous set of desired traits.
doi:10.1095/biolreprod.103.016147
PMCID: PMC2637358  PMID: 12700187
assisted reproductive technology; developmental biology; embryo; gene regulation
7.  Human metabolic profiles are stably controlled by genetic and environmental variation 
A comprehensive variation map of the human metabolome identifies genetic and stable-environmental sources as major drivers of metabolite concentrations. The data suggest that sample sizes of a few thousand are sufficient to detect metabolite biomarkers predictive of disease.
We designed a longitudinal twin study to characterize the genetic, stable-environmental, and longitudinally fluctuating influences on metabolite concentrations in two human biofluids—urine and plasma—focusing specifically on the representative subset of metabolites detectable by 1H nuclear magnetic resonance (1H NMR) spectroscopy.We identified widespread genetic and stable-environmental influences on the (urine and plasma) metabolomes, with (30 and 42%) attributable on average to familial sources, and (47 and 60%) attributable to longitudinally stable sources.Ten of the metabolites annotated in the study are estimated to have >60% familial contribution to their variation in concentration.Our findings have implications for the design and interpretation of 1H NMR-based molecular epidemiology studies. On the basis of the stable component of variation quantified in the current paper, we specified a model of disease association under which we inferred that sample sizes of a few thousand should be sufficient to detect disease-predictive metabolite biomarkers.
Metabolites are small molecules involved in biochemical processes in living systems. Their concentration in biofluids, such as urine and plasma, can offer insights into the functional status of biological pathways within an organism, and reflect input from multiple levels of biological organization—genetic, epigenetic, transcriptomic, and proteomic—as well as from environmental and lifestyle factors. Metabolite levels have the potential to indicate a broad variety of deviations from the ‘normal' physiological state, such as those that accompany a disease, or an increased susceptibility to disease. A number of recent studies have demonstrated that metabolite concentrations can be used to diagnose disease states accurately. A more ambitious goal is to identify metabolite biomarkers that are predictive of future disease onset, providing the possibility of intervention in susceptible individuals.
If an extreme concentration of a metabolite is to serve as an indicator of disease status, it is usually important to know the distribution of metabolite levels among healthy individuals. It is also useful to characterize the sources of that observed variation in the healthy population. A proportion of that variation—the heritable component—is attributable to genetic differences between individuals, potentially at many genetic loci. An effective, molecular indicator of a heritable, complex disease is likely to have a substantive heritable component. Non-heritable biological variation in metabolite concentrations can arise from a variety of environmental influences, such as dietary intake, lifestyle choices, general physical condition, composition of gut microflora, and use of medication. Variation across a population in stable-environmental influences leads to long-term differences between individuals in their baseline metabolite levels. Dynamic environmental pressures lead to short-term fluctuations within an individual about their baseline level. A metabolite whose concentration changes substantially in response to short-term pressures is relatively unlikely to offer long-term prediction of disease. In summary, the potential suitability of a metabolite to predict disease is reflected by the relative contributions of heritable and stable/unstable-environmental factors to its variation in concentration across the healthy population.
Studies involving twins are an established technique for quantifying the heritable component of phenotypes in human populations. Monozygotic (MZ) twins share the same DNA genome-wide, while dizygotic (DZ) twins share approximately half their inherited DNA, as do ordinary siblings. By comparing the average extent of phenotypic concordance within MZ pairs to that within DZ pairs, it is possible to quantify the heritability of a trait, and also to quantify the familiality, which refers to the combination of heritable and common-environmental effects (i.e., environmental influences shared by twins in a pair). In addition to incorporating twins into the study design, it is useful to quantify the phenotype in some individuals at multiple time points. The longitudinal aspect of such a study allows environmental effects to be decomposed into those that affect the phenotype over the short term and those that exert stable influence.
For the current study, urine and blood samples were collected from a cohort of MZ and DZ twins, with some twins donating samples on two occasions several months apart. Samples were analysed by 1H nuclear magnetic resonance (1H NMR) spectroscopy—an untargeted, discovery-driven technique for quantifying metabolite concentrations in biological samples. The application of 1H NMR to a biological sample creates a spectrum, made up of multiple peaks, with each peak's size quantitatively representing the concentration of its corresponding hydrogen-containing metabolite.
In each biological sample in our study, we extracted a full set of peaks, and thereby quantified the concentrations of all common plasma and urine metabolites detectable by 1H NMR. We developed bespoke statistical methods to decompose the observed concentration variation at each metabolite peak into that originating from familial, individual-environmental, and unstable-environmental sources.
We quantified the variability landscape across all common metabolite peaks in the urine and plasma 1H NMR metabolomes. We annotated a subset of peaks with a total of 65 metabolites; the variance decompositions for these are shown in Figure 1. Ten metabolites' concentrations were estimated to have familial contributions in excess of 60%. The average proportion of stable variation across all extracted metabolite peaks was estimated to be 47% in the urine samples and 60% in the plasma samples; the average estimated familiality was 30% for urine and 42% for plasma. These results comprise the first quantitative variation map of the 1H NMR metabolome. The identification and quantification of substantive widespread stability provides support for the use of these biofluids in molecular epidemiology studies. On the basis of our findings, we performed power calculations for a hypothetical study searching for predictive disease biomarkers among 1H NMR-detectable urine and plasma metabolites. Our calculations suggest that sample sizes of 2000–5000 should allow reliable identification of disease-predictive metabolite concentrations explaining 5–10% of disease risk, while greater sample sizes of 5000–20 000 would be required to identify metabolite concentrations explaining 1–2% of disease risk.
1H Nuclear Magnetic Resonance spectroscopy (1H NMR) is increasingly used to measure metabolite concentrations in sets of biological samples for top-down systems biology and molecular epidemiology. For such purposes, knowledge of the sources of human variation in metabolite concentrations is valuable, but currently sparse. We conducted and analysed a study to create such a resource. In our unique design, identical and non-identical twin pairs donated plasma and urine samples longitudinally. We acquired 1H NMR spectra on the samples, and statistically decomposed variation in metabolite concentration into familial (genetic and common-environmental), individual-environmental, and longitudinally unstable components. We estimate that stable variation, comprising familial and individual-environmental factors, accounts on average for 60% (plasma) and 47% (urine) of biological variation in 1H NMR-detectable metabolite concentrations. Clinically predictive metabolic variation is likely nested within this stable component, so our results have implications for the effective design of biomarker-discovery studies. We provide a power-calculation method which reveals that sample sizes of a few thousand should offer sufficient statistical precision to detect 1H NMR-based biomarkers quantifying predisposition to disease.
doi:10.1038/msb.2011.57
PMCID: PMC3202796  PMID: 21878913
biomarker; 1H nuclear magnetic resonance spectroscopy; metabolome-wide association study; top-down systems biology; variance decomposition
8.  Upper-Room Ultraviolet Light and Negative Air Ionization to Prevent Tuberculosis Transmission 
PLoS Medicine  2009;6(3):e1000043.
Background
Institutional tuberculosis (TB) transmission is an important public health problem highlighted by the HIV/AIDS pandemic and the emergence of multidrug- and extensively drug-resistant TB. Effective TB infection control measures are urgently needed. We evaluated the efficacy of upper-room ultraviolet (UV) lights and negative air ionization for preventing airborne TB transmission using a guinea pig air-sampling model to measure the TB infectiousness of ward air.
Methods and Findings
For 535 consecutive days, exhaust air from an HIV-TB ward in Lima, Perú, was passed through three guinea pig air-sampling enclosures each housing approximately 150 guinea pigs, using a 2-d cycle. On UV-off days, ward air passed in parallel through a control animal enclosure and a similar enclosure containing negative ionizers. On UV-on days, UV lights and mixing fans were turned on in the ward, and a third animal enclosure alone received ward air. TB infection in guinea pigs was defined by monthly tuberculin skin tests. All guinea pigs underwent autopsy to test for TB disease, defined by characteristic autopsy changes or by the culture of Mycobacterium tuberculosis from organs. 35% (106/304) of guinea pigs in the control group developed TB infection, and this was reduced to 14% (43/303) by ionizers, and to 9.5% (29/307) by UV lights (both p < 0.0001 compared with the control group). TB disease was confirmed in 8.6% (26/304) of control group animals, and this was reduced to 4.3% (13/303) by ionizers, and to 3.6% (11/307) by UV lights (both p < 0.03 compared with the control group). Time-to-event analysis demonstrated that TB infection was prevented by ionizers (log-rank 27; p < 0.0001) and by UV lights (log-rank 46; p < 0.0001). Time-to-event analysis also demonstrated that TB disease was prevented by ionizers (log-rank 3.7; p = 0.055) and by UV lights (log-rank 5.4; p = 0.02). An alternative analysis using an airborne infection model demonstrated that ionizers prevented 60% of TB infection and 51% of TB disease, and that UV lights prevented 70% of TB infection and 54% of TB disease. In all analysis strategies, UV lights tended to be more protective than ionizers.
Conclusions
Upper-room UV lights and negative air ionization each prevented most airborne TB transmission detectable by guinea pig air sampling. Provided there is adequate mixing of room air, upper-room UV light is an effective, low-cost intervention for use in TB infection control in high-risk clinical settings.
Using a guinea-pig detection model, Rod Escombe and colleagues find that upper-room UV lamps in hospital rooms can substantially reduce airborne transmission ofMycobacterium tuberculosis.
Editors' Summary
Background.
Tuberculosis—a contagious infection, usually of the lungs—kills nearly 2 million people annually. It is caused by Mycobacterium tuberculosis, bacteria that are spread in airborne droplets when people with tuberculosis cough or sneeze. Most people infected with M. tuberculosis do not become ill—their immune system contains the infection. However, the bacteria remain dormant within the body and can cause disease years later if immunity declines because of, for example, infection with human immunodeficiency virus (HIV), the cause of acquired immunodeficiency syndrome (AIDS). The symptoms of tuberculosis include a persistent cough, weight loss, and night sweats. Infection with M. tuberculosis is diagnosed using the tuberculin skin test. Tests for tuberculosis itself include chest X-rays and sputum cultures (in which bacteriologists try to grow M. tuberculosis from mucus brought up from the lungs by coughing). Tuberculosis can usually be cured by taking several powerful antibiotics daily for several months. Drug-resistant tuberculosis is much harder to cure, requiring multiple second-line antibiotics for up to two years or more. Tuberculosis transmission can be reduced by, for example, hospitalizing people with tuberculosis in isolation wards in which negative-pressure mechanical ventilation is used to reduce the concentration of infectious airborne droplets.
Why Was This Study Done?
After the development of antibiotics capable of killing M. tuberculosis in the mid 20th century, it seemed that tuberculosis would become a disease of the past. But in the mid 1980s, drug-resistant M. tuberculosis strains began to emerge, the HIV/AIDS epidemic took hold, and tuberculosis resurged to today's worrying levels. New ways of reducing tuberculosis transmission, particularly in health care settings and in resource-limited settings, are now urgently needed. The need for effective infection control measures is especially urgent in HIV care programs where highly susceptible individuals frequently mix with people with tuberculosis. In this study, the researchers use a guinea pig air-sampling model (which was first used in the 1950s to show that tuberculosis is an airborne infection) to investigate whether upper-room ultraviolet (UV) lights in patient rooms and negative air ionization can prevent airborne tuberculosis transmission. UV light kills M. tuberculosis; negative ionization gives airborne particles a charge that makes them stick to surfaces.
What Did the Researchers Do and Find?
The researchers exposed a group of control guinea pigs kept in a special air-sampling enclosure to untreated air from an HIV–TB ward in Lima (Perú). Another group of animals (the UV group) breathed air from the same ward, but only on the days that UV lights suspended near the ward's ceiling were turned on, together with mixing fans to mix up the room air. The “ionizer group” had a negative ionizer switched on in their enclosure when they were exposed to ward air (each group of animals was exposed to ward air every other day). The animals were tested monthly with the tuberculin skin test and all were examined for tuberculosis disease when they became infected with tuberculosis or at the end of the 535-day experiment. 35% of the control animals, 14% of the ionizer group animals, and 9.5% of the UV group animals developed M. tuberculosis infections. Tuberculosis disease was found in 8.6% of the control animals but in only 4.3% and 3.6% of the animals in the ionizer and UV groups, respectively. A “time-to-event analysis” also showed that UV lights and ionizers reduced tuberculosis infection and disease. Finally, an analysis of the data using an airborne infection model indicated that ionizers and UV lights prevented 60% and 70% of tuberculosis infections, respectively.
What Do These Findings Mean?
These findings indicate that upper-room UV lights, combined with adequate air mixing, or negative air ionization with special large-scale ionizers can prevent most airborne tuberculosis transmission to guinea pigs exposed to hospital room air. The effectiveness of these approaches in reducing tuberculosis transmission between people is likely to be similar, although remains to be tested. Nevertheless, this first study of the effect of upper-air UV light and of negative air ionization on airborne transmission in a clinical setting suggests that both approaches could be potentially important tuberculosis infection control measures. Furthermore, the UV light approach might provide a relatively low-cost intervention for possible use in waiting rooms and other overcrowded settings where patients with undiagnosed, untreated tuberculosis—individuals who tend to be highly infectious—are likely to come into contact with other susceptible patients, health care workers, and visitors.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000043.
The US National Institute of Allergy and Infectious Diseases provides information on all aspects of tuberculosis, including multidrug-resistance tuberculosis, and on tuberculosis and HIV
The US Centers for Disease Control and Prevention provide several fact sheets and other information resources about all aspects of tuberculosis, including Guidelines for preventing the Transmission of Mycobacterium tuberculosis in Health-Care Settings, 2005 (some information in Spanish is also available)
The World Health Organization's 2008 report “Global Tuberculosis Control—Surveillance, Planning, Financing” provides a snapshot of the current state of the global tuberculosis epidemic and links to information about all aspects of tuberculosis and its control (in several languages)
Tuberculosis Infection-Control in the Era of Expanding HIV Care and Treatment is another report from the World Health Organization
HIVInsite provides detailed information about the combination of HIV infection and tuberculosis
Avert, an international AIDS charity, also provides information about the interaction between HIV and tuberculosis
GHD (Global Health Delivery) Online is an online resource dedicated to TB infection control, and is moderated by world experts
doi:10.1371/journal.pmed.1000043
PMCID: PMC2656548  PMID: 19296717
9.  Production of Transgenic Pigs with an Introduced Missense Mutation of the Bone Morphogenetic Protein Receptor Type IB Gene Related to Prolificacy 
In the last few decades, transgenic animal technology has witnessed an increasingly wide application in animal breeding. Reproductive traits are economically important to the pig industry. It has been shown that the bone morphogenetic protein receptor type IB (BMPR1B) A746G polymorphism is responsible for the fertility in sheep. However, this causal mutation exits exclusively in sheep and goat. In this study, we attempted to create transgenic pigs by introducing this mutation with the aim to improve reproductive traits in pigs. We successfully constructed a vector containing porcine BMPR1B coding sequence (CDS) with the mutant G allele of A746G mutation. In total, we obtained 24 cloned male piglets using handmade cloning (HMC) technique, and 12 individuals survived till maturation. A set of polymerase chain reactions indicated that 11 of 12 matured boars were transgene-positive individuals, and that the transgenic vector was most likely disrupted during cloning. Of 11 positive pigs, one (No. 11) lost a part of the terminator region but had the intact promoter and the CDS regions. cDNA sequencing showed that the introduced allele (746G) was expressed in multiple tissues of transgene-positive offspring of No.11. Western blot analysis revealed that BMPR1B protein expression in multiple tissues of transgene-positive F1 piglets was 0.5 to 2-fold higher than that in the transgene-negative siblings. The No. 11 boar showed normal litter size performance as normal pigs from the same breed. Transgene-positive F1 boars produced by No. 11 had higher semen volume, sperm concentration and total sperm per ejaculate than the negative siblings, although the differences did not reached statistical significance. Transgene-positive F1 sows had similar litter size performance to the negative siblings, and more data are needed to adequately assess the litter size performance. In conclusion, we obtained 24 cloned transgenic pigs with the modified porcine BMPR1B CDS using HMC. cDNA sequencing and western blot indicated that the exogenous BMPR1B CDS was successfully expressed in host pigs. The transgenic pigs showed normal litter size performance. However, no significant differences in litter size were found between transgene-positive and negative sows. Our study provides new insight into producing cloned transgenic livestock related to reproductive traits.
doi:10.5713/ajas.15.0505
PMCID: PMC4932586  PMID: 26954151
Transgenic; Handmade Cloning; Pig; BMPR1B; Coding Sequence; Reproductive Traits
10.  Development of Transgenic Cloned Pig Models of Skin Inflammation by DNA Transposon-Directed Ectopic Expression of Human β1 and α2 Integrin 
PLoS ONE  2012;7(5):e36658.
Integrins constitute a superfamily of transmembrane signaling receptors that play pivotal roles in cutaneous homeostasis by modulating cell growth and differentiation as well as inflammatory responses in the skin. Subrabasal expression of integrins α2 and/or β1 entails hyperproliferation and aberrant differentiation of keratinocytes and leads to dermal and epidermal influx of activated T-cells. The anatomical and physiological similarities between porcine and human skin make the pig a suitable model for human skin diseases. In efforts to generate a porcine model of cutaneous inflammation, we employed the Sleeping Beauty DNA transposon system for production of transgenic cloned Göttingen minipigs expressing human β1 or α2 integrin under the control of a promoter specific for subrabasal keratinocytes. Using pools of transgenic donor fibroblasts, cloning by somatic cell nuclear transfer was utilized to produce reconstructed embryos that were subsequently transferred to surrogate sows. The resulting pigs were all transgenic and harbored from one to six transgene integrants. Molecular analyses on skin biopsies and cultured keratinocytes showed ectopic expression of the human integrins and localization within the keratinocyte plasma membrane. Markers of perturbed skin homeostasis, including activation of the MAPK pathway, increased expression of the pro-inflammatory cytokine IL-1α, and enhanced expression of the transcription factor c-Fos, were identified in keratinocytes from β1 and α2 integrin-transgenic minipigs, suggesting the induction of a chronic inflammatory phenotype in the skin. Notably, cellular dysregulation obtained by overexpression of either β1 or α2 integrin occurred through different cellular signaling pathways. Our findings mark the creation of the first cloned pig models with molecular markers of skin inflammation. Despite the absence of an overt psoriatic phenotype, these animals may possess increased susceptibility to severe skin damage-induced inflammation and should be of great potential in studies aiming at the development and refinement of topical therapies for cutaneous inflammation including psoriasis.
doi:10.1371/journal.pone.0036658
PMCID: PMC3349713  PMID: 22590584
11.  A Phosphorylcholine-Containing Glycolipid-like Antigen Present on the Surface of Infective Stage Larvae of Ascaris spp. Is a Major Antibody Target in Infected Pigs and Humans 
PLoS Neglected Tropical Diseases  2016;10(12):e0005166.
Background
The pig parasite Ascaris suum plays and important role in veterinary medicine and represents a suitable model for A. lumbricoides, which infects over 800 million people. In pigs, continued exposure to Ascaris induces immunity at the level of the gut, protecting the host against migrating larvae. The objective of this study was to identify and characterize parasite antigens targeted by this local immune response that may be crucial for parasite invasion and establishment and to evaluate their protective and diagnostic potential.
Methodology/Principal Findings
Pigs were immunized by trickle infection for 30 weeks, challenged with 2,000 eggs at week 32 and euthanized two weeks after challenge. At necropsy, there was a 100% reduction in worms recovered from the intestine and a 97.2% reduction in liver white spots in comparison with challenged non-immune control animals. Antibodies purified from the intestinal mucus or from the supernatant of cultured antibody secreting cells from mesenteric lymph nodes of immune pigs were used to probe L3 extracts to identify antibody targets. This resulted in the recognition of a 12kDa antigen (As12) that is actively shed from infective Ascaris L3. As12 was characterized as a phosphorylcholine-containing glycolipid-like antigen that is highly resistant to different enzymatic and chemical treatments. Vaccinating pigs with an As12 fraction did not induce protective immunity to challenge infection. However, serological analysis using sera or plasma from experimentally infected pigs or naturally infected humans demonstrated that the As12 ELISA was able to detect long-term exposure to Ascaris with a high diagnostic sensitivity (98.4% and 92%, respectively) and specificity (95.5% and 90.0%) in pigs and humans, respectively.
Conclusions/Significance
These findings show the presence of a highly stage specific, glycolipid-like component (As12) that is actively secreted by infectious Ascaris larvae and which acts as a major antibody target in infected humans and pigs.
Author Summary
Roundworms infect millions of humans and pigs throughout the world. The pig roundworm A. suum is a good model for A. lumbricoides infection in humans due to similar host physiology and the close genetic relationship between the worms. The aim of this study was to identify and characterize early larval antigens that are targeted by antibodies at the level of the intestine in immune pigs and to evaluate their protective and diagnostic potential. In order to do so, we generated highly immune pigs by repeatedly infecting them with A. suum for a long time (32 weeks). After necropsy, locally harvested antibodies from the gut were used to screen larval extracts. Hereby one particular antigen, named As12, was detected. It was characterized as a molecule of glycolipid nature that is presented on, and actively secreted from, the surface of infective larvae. Pigs immunized with this antigen are not protected from subsequent challenge infection. Experimentally infected pigs or naturally infected humans do however mount a significant serological antibody response to the antigen. These findings shed light on a glycolipid-like antigen (As12) that is secreted by infectious Ascaris larvae and is targeted by the immune system of infected humans and pigs.
doi:10.1371/journal.pntd.0005166
PMCID: PMC5131908  PMID: 27906979
12.  Immunogenicity and Pathogenicity of Chimeric Infectious DNA Clones of Pathogenic Porcine Circovirus Type 2 (PCV2) and Nonpathogenic PCV1 in Weanling Pigs 
Journal of Virology  2003;77(20):11232-11243.
Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS), whereas the ubiquitous porcine circovirus type 1 (PCV1) is nonpathogenic for pigs. We report here the construction and characterization of two chimeric infectious DNA clones of PCV1 and PCV2. The chimeric PCV1-2 clone contains the PCV2 capsid gene cloned in the backbone of the nonpathogenic PCV1 genome. A reciprocal chimeric PCV2-1 DNA clone was also constructed by replacing the PCV2 capsid gene with that of PCV1 in the backbone of the PCV2 genome. The PCV1, PCV2, and chimeric PCV1-2 and PCV2-1 DNA clones were all shown to be infectious in PK-15 cells, and their growth characteristics in vitro were determined and compared. To evaluate the immunogenicity and pathogenicity of the chimeric infectious DNA clones, 40 specific-pathogen-free (SPF) pigs were randomly assigned into five groups of eight pigs each. Group 1 pigs received phosphate-buffered saline as the negative control. Group 2 pigs were each injected in the superficial inguinal lymph nodes with 200 μg of the PCV1 infectious DNA clone. Group 3 pigs were each similarly injected with 200 μg of the PCV2 infectious DNA clone, group 4 pigs were each injected with 200 μg of the chimeric PCV1-2 infectious DNA clone, and group 5 pigs were each injected with 200 μg of the reciprocal chimeric PCV2-1 infectious DNA clone. As expected, seroconversion to antibodies to the PCV2 capsid antigen was detected in group 3 and group 4 pigs. Group 2 and 5 pigs all seroconverted to PCV1 antibody. Gross and microscopic lesions in various tissues of animals inoculated with the PCV2 infectious DNA clone were significantly more severe than those found in pigs inoculated with PCV1, chimeric PCV1-2, and reciprocal chimeric PCV2-1 infectious DNA clones. These data indicated that the chimeric PCV1-2 virus with the immunogenic ORF2 capsid gene of pathogenic PCV2 cloned into the nonpathogenic PCV1 genomic backbone induces a specific antibody response to the pathogenic PCV2 capsid antigen but is attenuated in pigs. Future studies are warranted to evaluate the usefulness of the chimeric PCV1-2 infectious DNA clone as a genetically engineered live-attenuated vaccine against PCV2 infection and PMWS.
doi:10.1128/JVI.77.20.11232-11243.2003
PMCID: PMC224998  PMID: 14512571
13.  A Chimeric Porcine Circovirus (PCV) with the Immunogenic Capsid Gene of the Pathogenic PCV Type 2 (PCV2) Cloned into the Genomic Backbone of the Nonpathogenic PCV1 Induces Protective Immunity against PCV2 Infection in Pigs 
Journal of Virology  2004;78(12):6297-6303.
Porcine circovirus type 2 (PCV2) is associated with postweaning multisystemic wasting syndrome in pigs, whereas PCV1 is nonpathogenic. We previously demonstrated that a chimeric PCV1-2 virus (with the immunogenic capsid gene of PCV2 cloned into the backbone of PCV1) induces an antibody response to the PCV2 capsid protein and is attenuated in pigs. Here, we report that the attenuated chimeric PCV1-2 induces protective immunity to wild-type PCV2 challenge in pigs. A total of 48 specific-pathogen-free piglets were randomly and equally assigned to four groups of 12 pigs each. Pigs in group 1 were vaccinated by intramuscular injection with 200 μg of the chimeric PCV1-2 infectious DNA clone. Pigs in group 2 were vaccinated by intralymphoid injection with 200 μg of a chimeric PCV1-2 infectious DNA clone. Pigs in group 3 were vaccinated by intramuscular injection with 103.5 50% tissue culture infective doses (TCID50) of the chimeric PCV1-2 live virus. Pigs in group 4 were not vaccinated and served as controls. By 42 days postvaccination (DPV), the majority of pigs had seroconverted to PCV2 capsid antibody. At 42 DPV, all pigs were challenged intranasally and intramuscularly with 2 × 104.5 TCID50 of a wild-type pathogenic PCV2 virus. By 21 days postchallenge (DPC), 9 out of the 12 group 4 pigs were viremic for PCV2. Vaccinated animals in groups 1 to 3 had no detectable PCV2 viremia after challenge. At 21 DPC the lymph nodes in the nonvaccinated pigs were larger (P < 0.05) than those of vaccinated pigs. The PCV2 genomic copy loads in lymph nodes were reduced (P < 0.0001) in vaccinated pigs. Moderate amounts of PCV2 antigen were detected in most lymphoid tissues of nonvaccinated pigs but in only 1 of 36 vaccinated pigs. Mild-to-severe lymphoid depletion and histiocytic replacement were detected in lymphoid tissues in the majority of nonvaccinated group 4 pigs but in only a few vaccinated group 1 to 3 pigs. The data from this study indicated that when given intramuscularly in pigs, the attenuated chimeric PCV1-2 live virus, as well as the chimeric PCV1-2 infectious DNA clone, induces protective immunity against PCV2 infection and could potentially serve as an effective vaccine.
doi:10.1128/JVI.78.12.6297-6303.2004
PMCID: PMC416547  PMID: 15163723
14.  Scabies Mites Alter the Skin Microbiome and Promote Growth of Opportunistic Pathogens in a Porcine Model 
Background
The resident skin microbiota plays an important role in restricting pathogenic bacteria, thereby protecting the host. Scabies mites (Sarcoptes scabiei) are thought to promote bacterial infections by breaching the skin barrier and excreting molecules that inhibit host innate immune responses. Epidemiological studies in humans confirm increased incidence of impetigo, generally caused by Staphylococcus aureus and Streptococcus pyogenes, secondary to the epidermal infestation with the parasitic mite. It is therefore possible that mite infestation could alter the healthy skin microbiota making way for the opportunistic pathogens. A longitudinal study to test this hypothesis in humans is near impossible due to ethical reasons. In a porcine model we generated scabies infestations closely resembling the disease manifestation in humans and investigated the scabies associated changes in the skin microbiota over the course of a mite infestation.
Methodology/Principal Findings
In a 21 week trial, skin scrapings were collected from pigs infected with S. scabies var. suis and scabies-free control animals. A total of 96 skin scrapings were collected before, during infection and after acaricide treatment, and analyzed by bacterial 16S rDNA tag-encoded FLX-titanium amplicon pyrosequencing. We found significant changes in the epidermal microbiota, in particular a dramatic increase in Staphylococcus correlating with the onset of mite infestation in animals challenged with scabies mites. This increase persisted beyond treatment from mite infection and healing of skin. Furthermore, the staphylococci population shifted from the commensal S. hominis on the healthy skin prior to scabies mite challenge to S. chromogenes, which is increasingly recognized as being pathogenic, coinciding with scabies infection in pigs. In contrast, all animals in the scabies-free cohort remained relatively free of Staphylococcus throughout the trial.
Conclusions/Significance
This is the first experimental in vivo evidence supporting previous assumptions that establishment of pathogens follow scabies infection. Our findings provide an explanation for a biologically important aspect of the disease pathogenesis. The methods developed from this pig trial will serve as a guide to analyze human clinical samples. Studies building on this will offer implications for development of novel intervention strategies against the mites and the secondary infections.
Author Summary
Scabies is a neglected, contagious skin disease caused by a parasitic mite Sarcoptes scabiei. It is highly prevalent world-wide, and now recognized as a possible underlying factor for secondary bacterial infections with potential serious downstream complications. There is currently few experimental data demonstrating directly that mite infestation promotes bacterial infections. Due to remarkable similarities in terms of immunology, physiology and skin anatomy between pigs and humans, we developed a sustainable porcine model enabling in vivo studies of scabies mite infestations. Here, we investigated the impact of the scabies mite infection on the normal pig skin microbiota in the inner ear pinnae in young piglets. Samples obtained prior to, during infection and after acaricide treatment were analyzed by sequencing of bacterial 16S rDNA. We report that scabies infestation has an impact on the host's skin microbiota. Staphylococcus abundance increased with the onset of infection and remained beyond treatment and healing. A shift from commensal to pathogenic Staphylococci was observed. This study supports the link between scabies and Staphylococcus infections, as seen in humans. It is the first in vivo demonstration of a mite induced shift in the skin microbiota, providing a basis for a similar study in humans.
doi:10.1371/journal.pntd.0002897
PMCID: PMC4038468  PMID: 24875186
15.  Genome-wide association analyses for meat quality traits in Chinese Erhualian pigs and a Western Duroc × (Landrace × Yorkshire) commercial population 
Background
Understanding the genetic mechanisms that underlie meat quality traits is essential to improve pork quality. To date, most quantitative trait loci (QTL) analyses have been performed on F2 crosses between outbred pig strains and have led to the identification of numerous QTL. However, because linkage disequilibrium is high in such crosses, QTL mapping precision is unsatisfactory and only a few QTL have been found to segregate within outbred strains, which limits their use to improve animal performance. To detect QTL in outbred pig populations of Chinese and Western origins, we performed genome-wide association studies (GWAS) for meat quality traits in Chinese purebred Erhualian pigs and a Western Duroc × (Landrace × Yorkshire) (DLY) commercial population.
Methods
Three hundred and thirty six Chinese Erhualian and 610 DLY pigs were genotyped using the Illumina PorcineSNP60K Beadchip and evaluated for 20 meat quality traits. After quality control, 35 985 and 56 216 single nucleotide polymorphisms (SNPs) were available for the Chinese Erhualian and DLY datasets, respectively, and were used to perform two separate GWAS. We also performed a meta-analysis that combined P-values and effects of 29 516 SNPs that were common to Erhualian, DLY, F2 and Sutai pig populations.
Results
We detected 28 and nine suggestive SNPs that surpassed the significance level for meat quality in Erhualian and DLY pigs, respectively. Among these SNPs, ss131261254 on pig chromosome 4 (SSC4) was the most significant (P = 7.97E-09) and was associated with drip loss in Erhualian pigs. Our results suggested that at least two QTL on SSC12 and on SSC15 may have pleiotropic effects on several related traits. All the QTL that were detected by GWAS were population-specific, including 12 novel regions. However, the meta-analysis revealed seven novel QTL for meat characteristics, which suggests the existence of common underlying variants that may differ in frequency across populations. These QTL regions contain several relevant candidate genes.
Conclusions
These findings provide valuable insights into the molecular basis of convergent evolution of meat quality traits in Chinese and Western breeds that show divergent phenotypes. They may contribute to genetic improvement of purebreds for crossbred performance.
Electronic supplementary material
The online version of this article (doi:10.1186/s12711-015-0120-x) contains supplementary material, which is available to authorized users.
doi:10.1186/s12711-015-0120-x
PMCID: PMC4427942  PMID: 25962760
16.  Cloned Genomic DNA of Type 2 Porcine Circovirus Is Infectious When Injected Directly into the Liver and Lymph Nodes of Pigs: Characterization of Clinical Disease, Virus Distribution, and Pathologic Lesions 
Journal of Virology  2002;76(2):541-551.
Infection of animals with a molecular viral clone is critical to study the genetic determinants of viral replication and virulence in the host. Type 2 porcine circovirus (PCV2) has been incriminated as the cause of postweaning multisystemic wasting syndrome (PMWS), an emerging disease in pigs. We report here for the first time the construction and use of an infectious molecular DNA clone of PCV2 to characterize the disease and pathologic lesions associated with PCV2 infection by direct in vivo transfection of pigs with the molecular clone. The PCV2 molecular clone was generated by ligating two copies of the complete PCV2 genome in tandem into the pBluescript SK (pSK) vector and was shown to be infectious in vitro when transfected into PK-15 cells. Forty specific-pathogen-free pigs at 4 weeks of age were randomly assigned to four groups of 10 each. Group 1 pigs served as uninoculated controls. Pigs in group 2 were each inoculated intranasally with about 1.9 × 105 50% tissue culture infective doses of a homogeneous PCV2 live virus stock derived from the molecular clone. Pigs in group 3 were each injected intrahepatically with 200 μg of the cloned PCV2 plasmid DNA, and pigs in group 4 were each injected into the superficial iliac lymph nodes with 200 μg of the cloned PCV2 plasmid DNA. Animals injected with the cloned PCV2 plasmid DNA developed infection resembling that induced by intranasal inoculation with PCV2 live virus stock. Seroconversion to PCV2-specific antibody was detected in the majority of pigs from the three inoculated groups at 35 days postinoculation (DPI). Viremia, beginning at 14 DPI and lasting 2 to 4 weeks, was detected in the majority of the pigs from all three inoculated groups. There were no remarkable clinical signs of PMWS in control or any of the inoculated pigs. Gross lesions in pigs of the three inoculated groups were similar and were characterized by systemically enlarged, tan lymph nodes and lungs that failed to collapse. Histopathological lesions and PCV2-specific antigen were detected in numerous tissues and organs, including brain, lung, heart, kidney, tonsil, lymph nodes, spleen, ileum, and liver of infected pigs. This study more definitively characterizes the clinical course and pathologic lesions exclusively attributable to PCV2 infection. The data from this study indicate that the cloned PCV2 genomic DNA may replace infectious virus for future PCV2 pathogenesis and immunization studies. The data also suggest that PCV2, although essential for development of PMWS, may require other factors or agents to induce the full spectrum of clinical signs and lesions associated with advanced cases of PMWS.
doi:10.1128/JVI.76.2.541-551.2002
PMCID: PMC136831  PMID: 11752145
17.  A Live-Attenuated Chimeric Porcine Circovirus Type 2 (PCV2) Vaccine Is Transmitted to Contact Pigs but Is Not Upregulated by Concurrent Infection with Porcine Parvovirus (PPV) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Is Efficacious in a PCV2b-PRRSV-PPV Challenge Model▿ 
The live chimeric porcine circovirus type 2 (PCV2) vaccine with the capsid gene of the emerging subtype 2b cloned in the genomic backbone of the nonpathogenic PCV1 is attenuated in vivo and induces protective immunity against PCV2. To further determine the safety and efficacy of this experimental vaccine, we tested for evidence of pig-to-pig transmission by commingling nonvaccinated and vaccinated pigs, determined potential upregulation by simultaneous vaccination and infection with porcine parvovirus (PPV) and porcine reproductive and respiratory syndrome virus (PRRSV), and determined vaccine efficacy by challenging pigs 4 weeks after vaccination with PCV2b, PRRSV, and PPV. Forty-six 21-day-old, PCV2-naïve pigs were randomly assigned to one of six groups. Twenty-nine of 46 pigs were challenged with PCV2b, PRRSV, and PPV at day 28, 8/46 remained nonvaccinated and nonchallenged and served as negative controls, and 9/46 remained nonchallenged and served as vaccination controls. All animals were necropsied at day 49. PCV1-PCV2 viremia was detected in nonvaccinated contact pigs commingled with vaccinated pigs, indicating pig-to-pig transmission; however, PCV1-PCV2 DNA levels remained low in all vaccinated and contact pigs regardless of concurrent infection. Finally, vaccination 28 days before challenge resulted in significantly (P < 0.05) decreased amounts of PCV2 in tissues and sera and significantly (P < 0.05) reduced macroscopic and microscopic lesions. The results of this study indicate that the experimental live-attenuated chimeric PCV2 vaccine, although transmissible to contact pigs, remains attenuated in pigs concurrently infected with PRRSV and PPV and induces protective immunity against PCV2b when it is administered 28 days before PCV2 exposure.
doi:10.1128/CVI.05057-11
PMCID: PMC3147358  PMID: 21653745
18.  Targeted mutations in myostatin by zinc-finger nucleases result in double-muscled phenotype in Meishan pigs 
Scientific Reports  2015;5:14435.
Myostatin (MSTN) is a dominant inhibitor of skeletal muscle development and growth. Mutations in MSTN gene can lead to muscle hypertrophy or double-muscled (DM) phenotype in cattle, sheep, dog and human. However, there has not been reported significant muscle phenotypes in pigs in association with MSTN mutations. Pigs are an important source of meat production, as well as serve as a preferred animal model for the studies of human disease. To study the impacts of MSTN mutations on skeletal muscle growth in pigs, we generated MSTN-mutant Meishan pigs with no marker gene via zinc finger nucleases (ZFN) technology. The MSTN-mutant pigs developed and grew normally, had increased muscle mass with decreased fat accumulation compared with wild type pigs, and homozygote MSTN mutant (MSTN−/−) pigs had apparent DM phenotype, and individual muscle mass increased by 100% over their wild-type controls (MSTN+/+) at eight months of age as a result of myofiber hyperplasia. Interestingly, 20% MSTN-mutant pigs had one extra thoracic vertebra. The MSTN-mutant pigs will not only offer a way of fast genetic improvement of lean meat for local fat-type indigenous pig breeds, but also serve as an important large animal model for biomedical studies of musculoskeletal formation, development and diseases.
doi:10.1038/srep14435
PMCID: PMC4585837  PMID: 26400270
19.  Plasma Proteome Profiles Associated with Diet-Induced Metabolic Syndrome and the Early Onset of Metabolic Syndrome in a Pig Model 
PLoS ONE  2013;8(9):e73087.
Obesity and related diabetes are important health threatening multifactorial metabolic diseases and it has been suggested that 25% of all diabetic patients are unaware of their patho-physiological condition. Biomarkers for monitoring and control are available, but early stage predictive biomarkers enabling prevention of these diseases are still lacking. We used the pig as a model to study metabolic disease because humans and pigs share a multitude of metabolic similarities. Diabetes was chemically induced and control and diabetic pigs were either fed a high unsaturated fat (Mediterranean) diet or a high saturated fat/cholesterol/sugar (cafeteria) diet. Physiological parameters related to fat metabolism and diabetes were measured. Diabetic pigs' plasma proteome profiles differed more between the two diets than control pigs plasma proteome profiles. The expression levels of several proteins correlated well with (patho)physiological parameters related to the fat metabolism (cholesterol, VLDL, LDL, NEFA) and diabetes (Glucose) and to the diet fed to the animals. Studying only the control pigs as a model for metabolic syndrome when fed the two diets showed correlations to the same parameters but now more focused on insulin, glucose and abdominal fat depot parameters. We conclude that proteomic profiles can be used as a biomarker to identify pigs with developing metabolic syndrome (prediabetes) and diabetes when fed a cafeteria diet. It could be developed into a potential biomarkers for the early recognition of metabolic diseases.
doi:10.1371/journal.pone.0073087
PMCID: PMC3781149  PMID: 24086269
20.  Pathogenesis of Lassa virus infection in guinea pigs. 
Infection and Immunity  1982;37(2):771-778.
A rodent model for human Lassa fever was developed which uses inbred (strain 13) and outbred (Hartley) guinea pigs. Strain 13 guinea pigs were uniformly susceptible to lethal infection by 2 or more PFU of Lassa virus strain Josiah. In contrast, no more than 30% of the Hartley guinea pigs died regardless of the virus dose. In lethally infected strain 13 guinea pigs, peak titers of virus (10(7) to 10(8) PFU) occurred in the spleen and lymph nodes at 8 to 9 days, in the salivary glands at 11 days, and in the lung at 14 to 16 days. Virus reached low titers (10(4) PFU) in the plasma and brain and intermediate titers in the liver, adrenal glands, kidney, pancreas, and heart. In moribund animals, the most consistent and severe histological lesion as an interstitial pneumonia. In contrast, the brain was only minimally involved. The immune response of lethally infected strain 13 guinea pigs, as measured by the indirect fluorescent antibody test, was detectable within 10 days of infection and was similar in timing and intensity to the fluorescent antibody test response of both lethally infected and surviving outbred animals. In contrast to the fluorescent antibody response, neutralizing antibody developed late in convalescence and was thus detected only in surviving outbred guinea pigs. The availability of a rodent model for human Lassa fever in uniformly susceptible strain 13 guinea pigs should facilitate detailed pathophysiological studies and efficacy testing of antiviral drugs, candidate vaccines, and immunotherapy regimens to develop control methods for this life-threatening disease in humans.
Images
PMCID: PMC347596  PMID: 6749685
21.  Creation of Functional Viruses from Non-Functional cDNA Clones Obtained from an RNA Virus Population by the Use of Ancestral Reconstruction 
PLoS ONE  2015;10(10):e0140912.
RNA viruses have the highest known mutation rates. Consequently it is likely that a high proportion of individual RNA virus genomes, isolated from an infected host, will contain lethal mutations and be non-functional. This is problematic if the aim is to clone and investigate high-fitness, functional cDNAs and may also pose problems for sequence-based analysis of viral evolution. To address these challenges we have performed a study of the evolution of classical swine fever virus (CSFV) using deep sequencing and analysis of 84 full-length cDNA clones, each representing individual genomes from a moderately virulent isolate. In addition to here being used as a model for RNA viruses generally, CSFV has high socioeconomic importance and remains a threat to animal welfare and pig production. We find that the majority of the investigated genomes are non-functional and only 12% produced infectious RNA transcripts. Full length sequencing of cDNA clones and deep sequencing of the parental population identified substitutions important for the observed phenotypes. The investigated cDNA clones were furthermore used as the basis for inferring the sequence of functional viruses. Since each unique clone must necessarily be the descendant of a functional ancestor, we hypothesized that it should be possible to produce functional clones by reconstructing ancestral sequences. To test this we used phylogenetic methods to infer two ancestral sequences, which were then reconstructed as cDNA clones. Viruses rescued from the reconstructed cDNAs were tested in cell culture and pigs. Both reconstructed ancestral genomes proved functional, and displayed distinct phenotypes in vitro and in vivo. We suggest that reconstruction of ancestral viruses is a useful tool for experimental and computational investigations of virulence and viral evolution. Importantly, ancestral reconstruction can be done even on the basis of a set of sequences that all correspond to non-functional variants.
doi:10.1371/journal.pone.0140912
PMCID: PMC4613144  PMID: 26485566
22.  Expression of Innate Immune Response Genes in Liver and Three Types of Adipose Tissue in Cloned Pigs 
Cellular Reprogramming  2012;14(5):407-417.
Abstract
The pig has been proposed as a relevant model for human obesity-induced inflammation, and cloning may improve the applicability of this model. We tested the assumptions that cloning would reduce interindividual variation in gene expression of innate immune factors and that their expression would remain unaffected by the cloning process. We investigated the expression of 40 innate immune factors by high-throughput quantitative real-time PCR in samples from liver, abdominal subcutaneous adipose tissue (SAT), visceral adipose tissue (VAT), and neck SAT in cloned pigs compared to normal outbred pigs.
The variation in gene expression was found to be similar for the two groups, and the expression of a small number of genes was significantly affected by cloning. In the VAT and abdominal SAT, six out of seven significantly differentially expressed genes were downregulated in the clones. In contrast, most differently expressed genes in both liver and neck SAT were upregulated (seven out of eight). Remarkably, acute phase proteins (APPs) dominated the upregulated genes in the liver, whereas APP expression was either unchanged or downregulated in abdominal SAT and VAT. The general conclusion from this work is that cloning leads to subtle changes in specific subsets of innate immune genes. Such changes, even if minor, may have phenotypic effects over time, e.g., in models of long-term inflammation related to obesity.
doi:10.1089/cell.2012.0026
PMCID: PMC3459008  PMID: 22928970
23.  Metabolomic Perfusate Analysis during Kidney Machine Perfusion: The Pig Provides an Appropriate Model for Human Studies 
PLoS ONE  2014;9(12):e114818.
Introduction
Hypothermic machine perfusion offers great promise in kidney transplantation and experimental studies are needed to establish the optimal conditions for this to occur. Pig kidneys are considered to be a good model for this purpose and share many properties with human organs. However it is not established whether the metabolism of pig kidneys in such hypothermic hypoxic conditions is comparable to human organs.
Methods
Standard criteria human (n = 12) and porcine (n = 10) kidneys underwent HMP using the LifePort Kidney Transporter 1.0 (Organ Recovery Systems) using KPS-1 solution. Perfusate was sampled at 45 minutes and 4 hours of perfusion and metabolomic analysis performed using 1-D 1H-NMR spectroscopy.
Results
There was no inter-species difference in the number of metabolites identified. Of the 30 metabolites analysed, 16 (53.3%) were present in comparable concentrations in the pig and human kidney perfusates. The rate of change of concentration for 3-Hydroxybutyrate was greater for human kidneys (p<0.001). For the other 29 metabolites (96.7%), there was no difference in the rate of change of concentration between pig and human samples.
Conclusions
Whilst there are some differences between pig and human kidneys during HMP they appear to be metabolically similar and the pig seems to be a valid model for human studies.
doi:10.1371/journal.pone.0114818
PMCID: PMC4264773  PMID: 25502759
24.  Factors Associated with the Prevalence of Circulating Antigens to Porcine Cysticercosis in Three Villages of Burkina Faso 
Background
Little is known about porcine cysticercosis in Burkina Faso. We conducted a pilot study to estimate the prevalence of antigens of Taenia solium cysticercosis and to identify associated factors in pigs of three villages in Burkina Faso, selected to represent different pig management practices: one village where pigs are allowed to roam freely (Batondo), one village where pigs are penned part of the time (Pabré) and one village with limited pig farming (Nyonyogo).
Methods/Principal Findings
A clustered random sampling design was used. Data on socio-demographic characteristics (source of drinking water, presence of latrines in the household, type and number of breeding animals) and pig management practices were collected using a standardized questionnaire. Blood samples were collected from one pig per household to determine the presence of antigens of the larval stages of T. solium by the B158/B60 Ag-ELISA. The associations between seropositivity and socio-demographic and pig management practices were estimated using logistic regression. Proportions of 32.5% (95% CI 25.4–40.3), 39.6% (31.9–47.8), and 0% of pigs, were found positive for the presence of circulating antigens of T. solium in Batondo, Pabré, and Nyonyogo, respectively. The results of the logistic regression analyses suggested that people acquire knowledge on porcine cysticercosis following the contamination of their animals. The presence of antigens in the pigs' sera was not associated with the absence of latrines in the household, the source of drinking water or the status of infection in humans but was associated with pig rearing practices during the rainy season.
Conclusions/Significance
The results suggest that education of pig farmers is urgently needed to reduce the prevalence of this infection.
Author Summary
Taenia solium cysticercosis is a neglected tropical infection transmitted between humans and pigs. This infection is particularly common in areas where sanitation, hygiene and pig management practices are poor, and can sometimes lead to epilepsy in humans. There is very little information about the importance of this infection in Burkina Faso, even though pork meat is widely consumed in many villages. We conducted a pilot study in three villages: two villages where pig rearing and pork consumption are common (Batondo and Pabré) but with different pig management practices, and one village with limited pig farming and pork consumption (Nyonyogo). Blood tests were done on pigs and information on pig raising was collected from farmers. Our study demonstrated that at least one third of pigs are infected with cysticercosis in villages where they are raised, and, particularly when pigs are left to roam some or all of the time. It also demonstrated that farmers may not be aware of this disease until one of their animals is found to be infected. Thus, the study concluded that there is an urgent need for improving education in order to control this tropical disease.
doi:10.1371/journal.pntd.0000927
PMCID: PMC3014946  PMID: 21245913
25.  Production of Pigs Expressing a Transgene under the Control of a Tetracycline-Inducible System 
PLoS ONE  2014;9(1):e86146.
Pigs are anatomically and physiologically closer to humans than other laboratory animals. Transgenic (TG) pigs are widely used as models of human diseases. The aim of this study was to produce pigs expressing a tetracycline (Tet)-inducible transgene. The Tet-on system was first tested in infected donor cells. Porcine fetal fibroblasts were infected with a universal doxycycline-inducible vector containing the target gene enhanced green fluorescent protein (eGFP). At 1 day after treatment with 1 µg/ml doxycycline, the fluorescence intensity of these cells was increased. Somatic cell nuclear transfer (SCNT) was then performed using these donor cells. The Tet-on system was then tested in the generated porcine SCNT-TG embryos. Of 4,951 porcine SCNT-TG embryos generated, 850 were cultured in the presence of 1 µg/ml doxycycline in vitro. All of these embryos expressed eGFP and 15 embryos developed to blastocyst stage. The remaining 4,101 embryos were transferred to thirty three surrogate pigs from which thirty eight cloned TG piglets were obtained. PCR analysis showed that the transgene was inserted into the genome of each of these piglets. Two TG fibroblast cell lines were established from these TG piglets, and these cells were used as donor cells for re-cloning. The re-cloned SCNT embryos expressed the eGFP transgene under the control of doxycycline. These data show that the expression of transgenes in cloned TG pigs can be regulated by the Tet-on/off systems.
doi:10.1371/journal.pone.0086146
PMCID: PMC3893280  PMID: 24454957

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