Bama Miniature Pigs Research Articles

Experimental infection of Bama miniature pigs with a highly virulent classical swine fever virus

BackgroundCurrently, larger domestic pigs are only animals widely used in vaccine evaluation and pathogenicity study of classical swine fever virus (CSFV). This study was aimed to create an alternative animal experimental infection model of CSFV.ResultsTwenty specific-pathogen-free Bama miniature pigs were randomly divided into two groups and rooms, infected and non-infected, and the pigs in the infected group were inoculated intramuscularly with 104, 105 or 106 TCID50 (median tissue culture infective dose) CSFV Shimen strain (n = 5 × 3) or left uninoculated to serve as in-contact pigs (n = 3). The uninfected control pigs (n = 2) were housed in a separate room. Clinical signs, body temperature, viraemia, tissue antigen distribution, pathological changes and seroconversion were monitored. Clinical signs were observed as early as 2 days post-inoculation (dpi) in all infected pigs (though mild in contact pigs), but not non-infected control pigs. All inoculated pigs showed viraemia by 6 dpi. The in-contact pigs showed lower levels of viraemia. At 10 dpi, seroconversion was noted in five of the 15 inoculated pigs. All inoculated or one in-contact pigs died by 15 dpi.ConclusionsThese results show that Bama miniature pigs support productive CSFV infection and display clinical signs and pathological changes consistent with CSFV infections observed in larger domestic pigs.

Investigation on the Microcirculation Effect of Local Application of Natural Hirudin on Porcine Random Skin Flap Venous Congestion

Medicinal leech therapy is commonly used to treat venous congestion by local application to skin flaps, which are frequently employed in re-construction surgery. In the present study, we investigated the effect of natural hirudin, which was applied locally to treat the venous congestion, in random skin flaps in a Guangxi Bama miniature pig model. The congestive random skin flaps were divided into 3 groups: Group A (control group that was locally applied with saline), Group B (locally applied with 20ATU natural hirudin per each flap), Group C (locally applied with 40ATU natural hirudin per each flap). The microcirculatory changes were observed by measuring myeloperoxidase (MPO), malondialdehyde (MDA) and superoxide dismutase (SOD). The level of MPO, MDA and SOD activities in the tissue in hirudin-treated groups was significantly different compared with control group (P < 0.05), but there was no significant difference between group B and group C. At 12 days, as compared to the control group, flap survival rate in hirudin-treated groups was significantly different (P < 0.05), but there was no significant difference between the groups receiving two different doses of hirudin. Natural hirudin can improve microcirculation by inhibiting oxygen free radical damage and also improve flap survival time.

Selection of reference genes for gene expression studies in PBMC from Bama miniature pig under heat stress

Heat stress decreases immune function and increases disease susceptibility in stressed animals, which are important factors for industry and public health. We investigated the molecular mechanisms underlying heat stress by profiling the expression of target genes involved in the cellular response in the blood of Bama miniature pigs ( Sus scrofa domestica) over 21 days with the use of quantitative RT-PCR (qRT-PCR). Reliable standards were established for the normalization of qRT-PCR. Six potential reference genes were ranked by their stability using the geNorm and NormFinder programs. Ribosomal protein L4 ( RPL4) and TATA-box-binding protein ( TBP) ranked as the two most stably expressed genes, except on day 21 when beta-2-microglobulin ( B2M) was the most stable. Glyceraldehyde-3-phosphate dehydrogenase ( GAPDH) and ribosomal RNA 18S ( 18SRNA) were discarded as reference genes due to their unstable expression patterns. When B2M and TBP genes were selected as standards in combination, rather than GAPDH, a significant upregulation in Toll-like receptor 2 ( TLR2) expression was observed after 21 consecutive days of heat stress. These findings suggest that selection of an ideal reference gene is a key step in comparison of transcription profiles in Bama miniature pigs.

Establishment and evaluation of models for small-for-size graft injury without veno-venous bypass in miniature pigs

Objective To establish small-for-size (SFS) graft injury models in miniature pigs with high standardization, reproducibility and similarity to clinical situation. Methods Ba-Ma miniature pigs were introduced in this study and orthotopic liver transplantations (OLTs) were performed in 12 pigs with 30% liver volume allogeneil grafts (small portion of right paramedian lobe, right lateral lobe and caudate lobe) without veno-venous bypass. The profiles of intra-operational hemodynamics and metabolism were investigated. Animals were observed for 7 days with daily serum biochemistry and coagulation function exam. The survival rate related to operation itself and the SFS grafts were respectively calculated as well as the graft regenerative ratio at post-operational day (POD) 7. Results Graft weight as a percentage of the recipient's native liver weight (GW/RLW) and the total body weight (GW/BW) were (28. 63±4. 42)% and (0. 73±0.06)%. The mean operation time, anhepatic phase, and the time of blockage of infra-hepatic IVC were (191. 7±14. 2) min, (28. 3±3. 6) min, and (45. 0±5. 8) min. The survival rate related to the operation itself and the SFS graft were 83. 33% (10/12) and 40% (4/10), and the graft regenerative ratio at POD7 was (278. 06±42. 95) %. Contrast to the remarkable increase of heart rate and serum potassium during anhepatic phase, the mean arterial pressure, central venous pressure, rectal temperature, PH value and buffer excess had a significant decrease (P<0.01) with a gradual recovery after reperfusioa Serum ALT, AST, PT, Cr, and TB were significantly increased with a peak level at POD1 for the former 4 and POD2 for TB, and then began to decrease and favorably recovered at POD7, but TB, PT, and AST levels were still high when compared to those of prereperfusion (P<0. 05). Conclusion This model of OLT performed with 30% liver volume graft without veno-venous bypass was an ideal large animal model for series studies related to SFS graft injury. Key words: Small-for-size liver transplantation; Miniature pigs; Veno-venous bypass; Small-for-size syndrome

Evaluation of the biocompatibility and mechanical properties of xenogeneic (porcine) extracellular matrix (ECM) scaffold for pelvic reconstruction

Xenogeneic (porcine) extracellular matrix (ECM) scaffolds have been suggested as ideal biomaterials for regeneration medicine; however, ECM prepared from different tissue sources has shown distinctive biological properties. Therefore, a comprehensive understanding of biological characteristics of different tissue-derived ECM is essential in the design of scaffolds for pelvic reconstruction. We compared the biological properties of ECM derived from different tissue sources of Bama miniature pigs as a pelvic biological patch in terms of histological structure, water absorption ability, biodegradation ability, mechanical properties, antimicrobial activity, and biocompatibility in vitro. Different ECM scaffolds have distinct structural differences, and all have good biocompatibility, and UBM exhibited better water uptake ability (above 500%), anti-biodegradation ability, mechanical properties, antimicrobial activity, and stem cell attachment properties than other tissue-derived ECM. Porcine UBM might serve as an ideal pelvic biological patch.

Escherichia coli O157:H7 EDL933 has a strong virulence to Bama miniature pigs by injection and fails to colonize to their gastrointestinal tracts.

Detection of Shiga toxin-producing Escherichia coli O157:H7 from commercially grown pigs has been reported. Furthermore, the E. coli O157:H7 colonized model of pig has been established and E. coli O157:H7 could be transmitted from infected donor pigs to naive pigs directly and indirectly. In the present study, we want to know whether any E. coli O157:H7 strain can colonize to the alimentary tract of pig and the virulence of E. coli O157:H7 to pig by injection. Bama miniature pig was infected withE. coli O157:H7 EDL933 strain orally, but the organism could not be recovered from the feces and did not cause any tissue damage. Nevertheless, this pathogen introduced serious clinical symptoms and pathological injuries by injection, especially the nervous system and the injected pig exhibited severe neurological symptoms, including synclonus tremens, ataxia, head-pressing and recumbency, etc. The pig did not excrete urine and feces and the abdomen became tympanous. These data suggested that only certain E. coli O157:H7 strains could colonize to the GIs of pigs involved mechanisms that related to various factors. However, the organism has strong virulence to pig by injection mode and it is a risky pathogen to human health. Key words: Escherichia coli O157:H7, bama miniature pig, colonization, inoculation, pathological injury.

Impacts of heat stress on baseline immune measures and a subset of T cells in Bama miniature pigs

Understanding the stress-associated mechanisms of immune system function and the increased susceptibility of livestock to disease is now more important than ever because the majority of emerging animal diseases have proven to be zoonotic diseases and, therefore, threaten public health. The objective of the present study was to explore the effect of a 21-day heat stress on total white blood cell (WBC) counts, differential counts, a subset of T cells, NK cytotoxicity and neutrophil phagocytosis in Bama miniature pigs aged approximately 3 to 4 months. Pigs were assigned to one of two treatment groups: stress (heat, n = 10) or control (no stress treatment, n = 10). The results showed that the number of total WBCs was decreased in heat stressed pigs at day 7 (P ≤ 0.05). The percentages of eosinophils and monocytes were significantly higher in stressed pigs than in control pigs at day 1 (P ≤ 0.05). The number of CD8 T cells, unlike the CD3 and CD4 T cell, was significantly increased in heat stressed pigs compared to control pigs at day 1 and day 14 (P ≤ 0.05). Consequently, the ratio of CD4 and CD8 T cells was reduced in stressed pigs compared to control pigs at day 14 (P ≤ 0.05). Neither neutrophil phagocytosis nor natural killer cytotoxicity differed between the treated and control pigs at any time. The results indicate that heat stress influences CD8 T cells and the ratio of CD4 and CD8 cells. This change may play an important role in immune suppression induced by high air temperature.

CYTOLOGICAL STUDIES ON INBRED RYE

The phylogenetic relationship of five species of pigs including Guizhou miniature pig, Bama pig, Xisuangbanna inbred pig, Rongchang Pig and Landrance was studied by RAPD analysis. Thirty-nine single polymorphic primers were selected out of 190 primers. The amplified fragments of thirty-nine primers were analysed by the RAPDistance package version 1.04 and the phylogenetic tree was constructed using NJ method. The results indicated as the following: three strains of miniature pigs have close phylogenetic relationship and the phylogenetic relationship between Guizhou miniature pig and Bama miniature pig was much closer. Landrance and Rongchang pig have close phylogenetic relationship too, but their phylogenetic relationship is far from the three strains of miniature pigs.

Mechanism of the Protective Effects of N-acetylcysteine on the Heart of Brain-Dead Ba-Ma Miniature Pigs

Severe stress response induced by brain death leads to a marked increase in the expression of inflammatory cytokines regulated by nuclear factor-kappaB (NF-kappaB). N-acetylcysteine may inhibit activation of the NF-kappaB pathway. This study examined the expression of NF-kappaB in the hearts of brain-dead Ba-Ma miniature pigs and the protection potential of N-acetylcysteine. Ba-Ma miniature pigs were randomized into 3 groups: control group (Group C), N-acetylcysteine-free group (Group B), and N-acetylcysteine treatment group (Group N). At 6, 12, and 24 hours after the initial brain death, serum cardiac troponin-T (cTnT), tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 were examined. Heart tissue was taken 24 hours after the initial brain death. Structural changes of the heart and the expression of NF-kappaB were analyzed. At 6 hours after the initial brain death, serum levels of cTnT, TNF-alpha, IL-1beta, and IL-6 in Groups B and N began to increase. Levels in Group B increased more dramatically than in Group N. At 24 hours, cardiocyte damage was documented, but the damage in Group N was less severe than that in Group B. The expression of NF-kappaB in Groups B and N increased, and expression in Group B increased more sharply than in Group N. N-acetylcysteine can alleviate both structural and functional injury of the heart during brain death, which might be related to the inhibition of NF-kappaB expression and decreasing release of inflammatory mediators.

Protective effects of affiliating portasystemic shunt on graft injury in small-for-size liver transplantation in miniature pigs

To evaluate the protective effects of affiliating portasystemic shunt on small-for-size graft in liver transplantation. Fifteen Chinese Bama miniature pigs were divided into three groups: group A (small-for-size liver transplantation), group B (distal splenorenal shunt + small-for-size liver transplantation), and group C (mesocaval H-shape shunt + small-for-size liver transplantation). Animals were followed up for 7 days with survival, dynamical liver function biochemical parameters, liver biopsies, portal venous pressure (PVP) and portal blood flow (PBF). Animal survivals were as follows: group A, 1/5, group B, 3/5 and group C, 5/5.Group A resulted in abnormal liver function parameters that were significantly ameliorated in group B and C. The histological examination of graft in group A displayed severe pathologic changes including hepatocyte vacuolar change or necrosis, sinusoidal congestion, parenchymal hemorrhage. Affiliating portasystemic shunt significantly alleviated graft injuries in group B and C. PVP rose and peaked up to 28.6 mm Hg (1 mm Hg = 0.133 kPa), PBF fluctuated after reperfusion in group A, but group B and C with affiliating portasystemic shunt showed significantly lower PVP and maintained rather stable PBF after reperfusion. There were also statistical differences in PVP or PBF between group B and C. Affiliating portasystemic shunt effectively might protect small-for-size graft from injuries after reperfusion.

Mechanisms and effects of biosynthesis and apoptosis in repair of full-thickness skin defect with collagen-chitosan dermal stent

To investigate biosynthetic and apoptotic mechanisms in repair of full thickness skin defect with collagen-chitosan porous scaffold transplantation, and to determinate differences between wound repair with the scaffold transplantation and scar healing without the scaffold transplantation. The full thickness skin defects were made on 10 Bama miniature pigs and the bilayer dermal equivalent (BDE) composed of collagen-chitosan porous scaffold and silicone membrane was transplanted on wounds. Surfaces of wounds were observed at 1, 2, and 3 weeks after the BDE transplantation, and so were done the wound repairs after epidermis had been grafted for 2 weeks on surface of the scaffold which had been transplanted on skin defect wounds for 2 weeks. At the same time, TGF-beta1 expressions, apoptosis and self collagen replacement of scaffolds in wounds were detected in situ by immunohistochemical staining, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) and picrosirius red polarized light. Wounds without scaffold transplantation were studied as control. 1) Wounds with the scaffold transplantation were different from granulation tissue. 2) The peak of TGF-beta1 expression in the scaffold wounds was from 1 to 2 weeks after BDE transplantation, and TGF-beta1 expressions decreased continuously from 3 to 4 weeks. TGF-beta1 expressions increased continuously in the control wounds from 1 to 3 weeks and decreased on 4 weeks. TGF-beta1 expressions in the scaffold wounds on 1st and 2nd week were significantly higher than those in the corresponding control wounds, whereas, TGF-beta1 expressions in the scaffold wounds on 3rd and 4th week were significantly lower than those in the corresponding control wounds. 3) Apoptosis increased continuously in the scaffold wounds from 2 to 4 weeks after BDE transplantation, and so did in the control wounds from 3 to 4 weeks. However, apoptosis signals in the scaffold wounds on 2nd, 3rd, and 4th week after BDE transplantation were significantly more than those in the corresponding control wounds, and there was no difference between apoptosis signals in the scaffold wounds on 1st week after BDE transplantation and those in the corresponding control wounds. 4) Observation by picrosirius red polarized light method: self collagen began to synthesize in the scaffold wounds on 1st week after BDE transplantation, and scaffolds had been replaced by self collagen from 2 to 3 weeks after BDE transplantation. Collagen-chitosan porous scaffold plays a very important role in wound healing of full thickness skin defect. The mechanisms of wound repair by dermal scaffold are different from those by granulation and scar healing. It has a good future in repairing skin defect.

PREPARATION OF COLLAGEN-SULFONATED CARBOXYMETHYL CHITOSAN/SILICONE MEMBRANE BILAYER DERMAL EQUIVALENT AND ITS APPLICATION IN THE TREATMENT OF SCALDS OF PORCINE SKIN

制备了一种胶原-磺化羧甲基壳聚糖/硅橡胶皮肤再生材料,并以小型猪为模型,考察了其对烫伤全层皮肤缺损的修复性能.首先合成了磺化羧甲基壳聚糖,并对其结构进行了表征.制备了胶原-磺化羧甲基壳聚糖多孔支架,采用扫描电子显微镜(SEM)研究了磺化羧甲基壳聚糖含量对支架微结构的影响.随着磺化羧甲基壳聚糖含量的增大,胶原-磺化羧甲基壳聚糖支架从纤维结构向片状结构转化,且支架的孔径相对变大.采用体外成纤维细胞培养实验证明胶原-磺化羧甲基壳聚糖支架无明显细胞毒性.进一步将胶原-磺化羧甲基壳聚糖支架与硅橡胶膜复合,构建具有双层结构的皮肤再生材料.以小型猪为模型,评价了其对深度烫伤创面的修复性能.大体观察和组织学分析结果显示,胶原-磺化羧甲基壳聚糖/硅橡胶皮肤再生材料具有更快的血管化性能,且经该材料处理的创面能有效支持薄自体皮片的移植成活,实现深度烫伤创面的全层修复.

Tissue distribution of CYP3A29 mRNA expression in Bama miniature pig by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR)

Pig or minipig cytochrome P450 (CYP) 3A isozymes have been proven to be a good model of human drug metabolism, and human CYP3A seems to be well modelled by pig or minipig CYP3A29.We studied CYP3A29 mRNA expression in ten tissues (liver, duodenum, kidney, adrenal gland, skin, brain, lung, testis, uterus and ovary) in different age groups of Bama miniature pig by real-time reverse transcriptase-polymerase chain reaction (RT-PCR).In adult pig (4 and 6 months old), the most relative expression by normalization to β-actin of CYP3A29 mRNAs of tissues is in liver; then in duodenum, skin and kidney. Values of CYP3A29 in the extrahepatic tissues except for duodenum are very low compared with that in liver.In new-borns the mean CYP3A29 expression in duodenum was higher than that in liver, and the expression in skin was very low while high in 40 days, 4 months and 6 months age groups. For different ages, the increased CYP3A29 expression in liver and duodenum was detected with age. The effective CYP3A29 induction in liver was also detected by rifampin oral intubation. Apparent interindividual variability of CYP3A29 mRNA expression in Bama miniature pigs was detected.This work will broaden the understanding of the physiological functions of CYP3A29 in miniature pig, and promote Bama miniature pig’s application in pharmacological and toxicological studies.

Effect and Mechanism of Intermittent Myocardial Ischemia Induced by Exercise on Coronary Collateral Formation

To hypothesize that appropriate intermittent myocardial ischemia induced by exercise could safely promote coronary collateral formation in the ischemic area through the increased expression of vascular endothelial growth factor (VEGF) and its receptor fetal liver kinase-1 (Flk-1). A balloon constrictor was surgically implanted in the first obtuse marginal coronary artery (OM1) of Guangxi BA-MA miniature pigs. The subjects were divided into three groups: sham-operated (SO), pure ischemia (PI), and exercise training (ET). Subjects in the ET group performed individualized treadmill programs with two episodes of exercise-induced ischemia for 8 wks; two preexercise episodes of pure ischemia induced by brief OM1 occlusion were also conducted. Only pure ischemia was induced in the PI group, and the SO group remained sedentary for the experimental period. VEGF and Flk-1 expression levels were measured by Western blot and real-time reverse transcription polymerase chain reaction analyses; capillary density by immunohistochemistry; relative myocardial blood flow by microspheres; and cardiac troponin I by enzyme-linked immunosorbent assay. The relative myocardial blood flow, VEGF, Flk-1, and capillary density in the ET group were statistically higher than those in the PI and SO groups. All parameters in the PI group were statistically higher than those in the SO group. There was no myocardial damage in the ET or PI groups by cardiac troponin I. Intermittent myocardial ischemia induced by exercise with optimal stimulation safely promotes coronary collateral formation through increased VEGF and Flk-1 expression in a porcine model.

In vitro and in vivo biological performance of collagen-chitosan/silicone membrane bilayer dermal equivalent

Skin loss or damage affects severely the life quality of human being and can even cause death in many cases. We report here a bilayer dermal equivalent (BDE) composed of collagen-chitosan porous scaffold and silicone membrane, which can effectively induce the regeneration of dermis in an animal model of full thickness skin loss. The in vitro biosecurity test showed that the BDE had no cytotoxicity, and no remarkable sensitization and irritability. In vitro cell culture proved that the BDE had good biocompatibility to support the proliferation of fibroblasts. Animal test was performed on Bama miniature pig skin. Gross view and histological sections found plenty of fibroblasts and extracellular matrix in the regenerative scaffold after transplantation of the BDE for 4 weeks. Immunohistochemistry results proved that the BDE has the ability to support the angiogenesis of the regenerated dermis. All these results indicate that the BDE might be a promising equivalent in treating dermal loss.

A SWINE MODEL FOR LONG‐TERM EVALUATION OF PROSTHETIC HEART VALVES

The objective of this study was to develop a porcine model of mitral valve replacement (MVR) for long-term evaluation of prosthetic heart valves. Sixteen 25-kg male Bama miniature pigs underwent MVR using St Jude Medical valve (21 mm). Each animal was allocated to an anticoagulation protocol after surgery (group I, s.c. heparin injection and warfarin (n = 8); group II, s.c. low-molecular-weight heparin and warfarin (n = 8)) and was followed for up to 20 weeks. Terminal studies were carried out on all animals having survived for more than 140 days or died. Fourteen animals survived for more than 1 month without signs of heart failure. One of group I animals died from haemorrhagic (haemopericardium) complications on the 9th postoperative day, and another animal of group I died on the 18th postoperative day because of valve thrombosis. Compared with other species, humans and pigs show remarkable anatomical and physiological similarities. This model is promising for long-term preclinical evaluation of prosthetic heart valves and evaluation of postoperative anticoagulant agents.

Expression of CYP3A29 mRNA in Chinese experimental miniature pig’s livers quantitated with real-time reverse transcription polymerase chain reaction

CYP3A29 is the most important key enzyme for drug metabolism in pig's liver. Research of the characters of CYP3A29 mRNA expression in Chinese experimental miniature pig's livers is significant for evaluating it whether suitable for experimental model for drug metabolism mediated by human CYP3A4 enzyme. In this study, the levels of CYP3A29 mRNA expression in livers of Bama miniature pigs, Guizhou miniature pigs and Rongchang pigs were studied by TaqMan-mediated quantitative RT-PCR. Results indicated that the CYP3A29 mRNA expression levels in livers of these species were close to literatures of human. Levels of CYP3A29 mRNA expression were similar among livers of Bama miniature pigs, Guizhou miniature pigs and Rongchang pigs, but interindividual variations were quite large. It was suggested that Bama miniature pigs and Guizhou miniature pigs were both feasible for experimental animal model for evaluating drug metabolism in some degree.

Effects of NO-1886 on inflammation-associated cytokines in high-fat/high-sucrose/high-cholesterol diet-fed miniature pigs

Inflammation, closely associated with obesity, is emerging as an important risk factor for the pathophysiological development of atherosclerosis and diabetes mellitus. Fat balance is critical in the aetiology of obesity. Lipoprotein lipase is an important enzyme in lipid metabolism. The aim of this study was to investigate the long-term effect of the lipoprotein lipase activator, NO-1886, on inflammation cytokines, adiposity and related diseases in miniature pigs fed a high-fat/high-sucrose/high-cholesterol diet (HFSC diet). Chinese Bama-miniature pigs were fed a control diet or HFSC diet with or without NO-1886 for 5 months. The levels of inflammation-associated cytokines were determined using the antibody arrays. Feeding of the HFSC diet to miniature pigs markedly increased the expression of inflammatory cytokines. On the other hand, supplementation of NO-1886 to HFSC diet decreased the expression of inflammatory cytokines significantly, protecting against the development of atherosclerosis and diabetes mellitus. NO-1886 may have a beneficial effect on the most inflammation-associated cytokines, and this effect may contribute to improving atherosclerosis and diabetes mellitus.

The Degree of Inbreeding in 10 Chinese Indigenous Pig Breeds

The population inbreeding coefficients of 10 indigenous pig breeds were estimated by microsatellite markers. Results showed that: the inbreeding coefficient of Guizhou miniature pig was the highest (0.1992), and that of Hanzhong black pig the lowest (0.0727) among random mating populations. F test showed no significant difference in inbreeding coefficient among the 8 breeds (P >0.05). The inbreeding coefficient of the Guizhou miniature pig and Bama miniature pig inbreeding lines was 0.5907 and 0.4761, respectively.