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PubMed2003

Primary cultures of female swine genital epithelial cells in vitro: a new approach for the study of hormonal modulation of Chlamydia infection.

Previous studies have demonstrated that female reproductive hormones influence chlamydial infection both in vivo and in vitro. Due to the reduced availability of human genital tissues for research purposes, an alternative hormone-responsive model system was sought to study chlamydial pathogenesis. Mature female swine eliminated from breeding programs were selected as the animals of choice because of the similarity of a sexually transmitted disease syndrome and sequelae in swine to a disease syndrome and sequelae found in humans, because of the near identity of a natural infectious chlamydial isolate from swine to Chlamydia trachomatis serovar D from humans, and because a pig's epithelial cell physiology and the mean length of its estrous cycle are similar to those in humans. Epithelial cells from the cervix, uterus, and horns of the uterus were isolated, cultivated in vitro in Dulbecco's minimum essential medium-Hanks' F-12 (DMEM-F-12) medium with and without exogenous hormone supplementation, and analyzed for Chlamydia suis S-45 infectivity. The distribution of chlamydial inclusions in swine epithelial cells was uneven and was influenced by the genital tract site and hormone status. This study confirmed that, like primary human endometrial epithelial cells, estrogen-dominant swine epithelial cells are more susceptible to chlamydial infection than are progesterone-dominant cells. Further, the more differentiated luminal epithelial cells were more susceptible to infection than were glandular epithelial cells. Interestingly, chlamydial growth in mature luminal epithelia was morphologically more active than in glandular epithelia, where persistent chlamydial forms predominated. Attempts to reprogram epithelial cell physiology and thereby susceptibility to chlamydial infection by reverse-stage, exogenous hormonal supplementation were unsuccessful. Freshly isolated primary pig epithelial cells frozen at -80 degrees C in DMEM-F-12 medium with 10% dimethyl sulfoxide for several weeks can, after thawing, reform characteristic polarized monolayers in 3 to 5 days. Thus, primary swine genital epithelia cultured ex vivo appear to be an excellent cell model for dissecting the hormonal modulation of several aspects of chlamydial pathogenesis and infection.

Animals↗

PubMed2000

Positive human health effects of wearing a respirator in a swine barn.

STUDY OBJECTIVES: A study was conducted to evaluate the acute health effects of wearing an N-95 disposable respirator in a swine confinement facility. DESIGN: A crossover trial design was used in the study. SETTING: The study was carried out at the research facilities of the Centre for Agricultural Medicine, the Royal University Hospital, and the Prairie Swine Centre Inc, Saskatoon, Saskatchewan, Canada. PARTICIPANTS: Twenty-one nonsmoking healthy male subjects with no previous swine barn exposure participated in the study. INTERVENTIONS: The subjects participated in a laboratory session (baseline day), a 4-h exposure in a traditional swine room wearing the respirator (intervention day), and a 4-hour exposure in a traditional swine room without a respirator (nonintervention day). MEASUREMENTS: Lung function, methacholine challenge tests, blood counts, nasal lavage, and cytokines in serum and nasal lavage fluid. RESULTS: Mean (+/- SE) shift change in FEV(1), from preexposure to postexposure, was highest on nonintervention day (-8.1+/-1.01%) and was significantly different from intervention day (0.32+/-0.62%; p<0.0001) and baseline day (1.57+/-0.51%; p<0.0001). Similar patterns were observed in the mean values of the provocative concentration of a substance (methacholine) causing a 20% fall in FEV(1) (nonintervention day, 130.4+/-36.9 mg/mL; intervention day, 242.0+/-38.0 mg/mL; and baseline day, 328.0 mg/mL +/-34.1 mg/mL). Significant increases in serum neutrophil levels and nasal cell counts were observed on the nonintervention day in comparison to the baseline and intervention days. Significant increases also were found in the levels of cytokines interleukin (IL)-6 and IL-8 in nasal lavage fluid and in the levels of IL-6 in serum for the nonintervention day in comparison to the other 2 days. CONCLUSIONS: The results demonstrate that an N-95 disposable respirator can help to significantly reduce acute negative health effects in subjects not previously exposed to a swine barn environment.

Adolescent↗

PubMed2003

Swine HEV infection in south India and phylogenetic analysis (1985-1999).

Hepatitis E is endemic in India. It was recently noted that although all the Indian human hepatitis E virus (HEV) isolates (1976-2001) were placed in genotype I, the swine HEV recovered from western India (2000) belonged to genotype IV. This was in contrast to reports from the United States and Taiwan wherein both human and swine HEV belonged to the same genotype, i.e., genotypes III and IV, respectively. In order to validate these findings further, we retrospectively examined serum samples collected from pigs from southern India. Sequential serum samples from 45 (1985-1987) and 12 (1999) pigs from Karnataka state, south India, were screened for the presence of HEV RNA (nested PCR) and IgG-anti-HEV (ELISA). PCR products (Open Reading Frame-2 region) were sequenced and subjected to phylogenetic analysis. In this study, 42/45 (1985-1987) and 12/12 (1999) pigs showed seroconversion to IgG anti-HEV antibodies, with a mean age at seroconversion of 4.8 +/- 1.6 months. Four samples collected in 1999 and two samples collected during 1985 were HEV RNA positive. All swine HEV sequences clustered with genotype IV, demonstrating that swine HEV was prevalent among south Indian pigs for at least for 16 years and, similar to western India, belonged to genotype IV. Thus, genotype I and IV HEV continue to circulate in humans and pigs, respectively, from India. Whether swine HEV infects humans remains to be determined.

Animals↗

PubMed1999

Replication of African swine fever virus DNA in infected cells.

We have examined the ultrastructural localization of African swine fever virus DNA in thin-sections of infected cells by in situ hybridization and autoradiography. Virus-specific DNA sequences were found in the nucleus of infected Vero cells at early times in the synthesis of the viral DNA, forming dense foci localized in proximity to the nuclear membrane. At later times, the viral DNA was found exclusively in the cytoplasm. Electron microscopic autoradiography of African swine fever virus-infected macrophages showed that the nucleus is also a site of viral DNA replication at early times. These results provide further evidence of the existence of nuclear and cytoplasmic stages in the synthesis of African swine fever virus DNA. On the other hand, alkaline sucrose sedimentation analysis of the replicative intermediates synthesized in the nucleus and cytoplasm of infected macrophages showed that small DNA fragments ( approximately 6-12S) were synthesized in the nucleus at an early time, whereas at later times, larger fragments of approximately 37-49S were labeled in the cytoplasm. Pulse-chase experiments demonstrated that these fragments are precursors of the mature cross-linked viral DNA. The formation of dimeric concatemers, which are predominantly head-to-head linked, was observed by pulsed-field electrophoresis and restriction enzyme analysis at intermediate and late times in the replication of African swine fever virus DNA. Our findings suggest that the replication of African swine fever virus DNA proceeds by a de novo start mechanism with the synthesis of small DNA fragments, which are then converted into larger size molecules. Ligation or further elongation of these molecules would originate a two-unit concatemer with dimeric ends that could be resolved to generate the genomic DNA by site-specific nicking, rearrangement, and ligation as has been proposed in the de novo start model of Baroudy et al. (B. M. Baroudy, S. Venkatesam, and B. Moss, 1982, Cold Spring Harbor Symp. Quant. Biol. 47, 723-729) for the replication of vaccinia virus DNA.

African Swine Fever Virus↗

PubMed1995

Involvement of endogenous adenosine in ischaemic preconditioning in swine.

Adenosine release and the subsequent activation of adenosine receptors are involved in ischaemic preconditioning in dogs and rabbits. In the present study, we investigated whether adenosine also mediates ischaemic preconditioning in swine. Swine were used since, due to the lack of an innate collateral circulation, infarct development in this species most closely resembles that observed in humans. In 36 enflurane-anaesthetized swine the impact of increased adenosine breakdown with exogenous porcine adenosine deaminase (5 IU/ml blood/min) on global and regional myocardial function (sonomicrometry), subendocardial blood flow (ENDO, microspheres) and infarct size (IS, triphenyl tetrazolium chloride staining following 90 min ischaemia and 120 min reperfusion) were analysed. Low-flow ischaemia for 90 min at an ENDO of 0.09 +/- 0.04 (mean +/- SD) ml/min/g caused an IS of 13.2 +/- 9.7% (n = 8) of the area at risk. Ischaemic preconditioning by a cycle of 10 min low-flow ischaemia followed by 15 min reperfusion prior to the 90-min ischaemic period (ENDO = 0.06 +/- 0.03 ml/min/g) reduced IS to 2.6 +/- 3.0% (n = 11, P < 0.05). The interstitial adenosine concentration (microdialysis) increased from 1.60 +/- 0.87 nmol/ml to above 10 microM during ischaemia; with intracoronary adenosine deaminase, the interstitial adenosine concentration fell from 1.65 +/- 0.23 to 0.12 +/- 0.07 nmol/ml and did not increase during ischaemia. Adenosine deaminase per se did not alter IS after 90 min ischaemia (n = 7, ENDO = 0.08 +/- 0.04 ml/min/g, IS = 12.1 +/- 6.9%) but abolished the beneficial effect of ischaemic preconditioning (n = 10, ENDO = 0.06 +/- 0.03 ml/min/g, IS = 8.8 +/- 5.8%). For any given ENDO, IS was significantly reduced in the ischaemic preconditioned group compared with the other three groups. Global and regional myocardial function were comparable among all groups of swine. We conclude that endogenous adenosine mediates ischaemic preconditioning also in swine.

Adenosine↗

PubMed2006

Effects of gradual coronary artery occlusion and exercise training on gene expression in swine heart.

Gradual occlusion (O) of the swine left circumflex coronary artery (LCX) with an ameroid occluder results in complete O within 3 weeks, collateral vessel development, and compensatory hypertrophy. The purpose of this investigation was to determine the independent and combined effects of O and exercise training (E) on gene expression in the swine heart. Adult Yucatan miniature swine were assigned to one of the following groups (n=6-9/group): sedentary control (S), exercise-trained (E), sedentary swine subjected to LCX occlusion (SO), and exercise-trained swine with LCX occlusion (EO). Exercise consisted of progressive treadmill running conducted 5 d/wk for 16 weeks. Gene expression was studied in myocardium isolated from the collateral-dependent left ventricle free wall (LV) and the collateral-independent septum (SEP) by RNA blotting. E and O each stimulated cardiac hypertrophy independently (p<0.001) with no interaction. O but not E increased atrial natriuretic factor expression in the LV, but not in the SEP. E decreased the expression of beta-myosin heavy chain in the LV, but not in the SEP. E retarded the expression of collagen III mRNA in SEP; but not in the LV. Exercise training and coronary artery occlusion each stimulate cardiac hypertrophy independently and induce different patterns of gene expression.

Animals↗

PubMed1986

Comparison of experimental hypercholesterolemia and atherosclerosis in Göttingen mini-pigs and Swedish domestic swine.

Mini-pigs of the Göttingen strain and domestic swine of the Swedish Landrace were compared with respect to their susceptibility to alimentary hypercholesterolemia and experimental atherosclerosis. Hypercholesterolemia was produced in both strains by adding 11.2% egg yolk and 0.5% cholesterol to the diet. The plasma cholesterol rose significantly faster and reached a higher level in the mini-pigs than in the domestic swine. In both strains most of the elevated plasma cholesterol was found in the low density lipoprotein fraction. The basal plasma triglyceride level was higher in the mini-pigs and increased transiently in this strain when the lipid-rich diet was given. After 18 months of hypercholesterolemia all animals were killed and the aorta and the coronary arteries were examined for atherosclerotic lesions. The arterial content of free and esterified cholesterol, triglycerides and phospholipids were determined. From colour photographs of the aorta the percentage area of visible atherosclerosis in the abdominal part was calculated. Coronary arteries from both strains were sectioned and stained for microscopic investigation. Atherosclerotic lesions were found in the abdominal aorta in both strains and in the coronary arteries in the mini-pigs. In the coronary arteries from domestic swine no macroscopically visible atherosclerosis was found and the content of all arterial lipids was lower than in the mini-pigs. Microscopically, the coronary arteries from the mini-pigs showed pronounced intimal proliferative atheromatous lesions, whereas no atheromas were observed in the domestic swine. In the thoracic and abdominal aorta, however, there were no differences between the two strains, either with regard to the cholesterol and phospholipid content or in the atherosclerotic area. The content of triglycerides was somewhat higher in the abdominal aorta from mini-pigs than in that from domestic swine.

Animals↗

PubMed1992

The effect of exposure to a delta cya/delta crp mutant of Salmonella typhimurium on the subsequent colonization of swine by the wild-type parent strain.

This study characterizes the clinical response and colonization pattern of caesarean-derived, colostrum-deprived swine exposed to a delta cya/delta crp mutant (chi 4233) of S. typhimurium and challenged with the wild-type parent strain. chi 4233 was mildly virulent in swine and induced transient fever and soft stools. Chi 4233 colonized the ileum, cecum, liver, spleen, tonsils, and mandibular and ileocolic lymph nodes of swine in a manner similar to the parental wild-type, but the numbers of S. typhimurium (chi 4233) in the ileum were 100- to 1000-fold less than those of pigs exposed to the parental wild-type. Pigs exposed to chi 4233 21 days before parental wild-type challenge demonstrated a milder clinical response to challenge than did pigs that did not receive chi 4233. The wild-type populations in the ilea of chi 4233-exposed pigs after challenge were 100- to 10,000-fold less than those in pigs not receiving chi 4233. The liver, spleen, and ileocolic lymph nodes were cleared of wild-type S. typhimurium more quickly after challenge in chi 4233-exposed pigs. The populations of chi 4233 in the ilea of exposed pigs after wild-type challenge were also less than would have been expected in unchallenged pigs. Thus, exposure of swine to a delta cya/delta crp mutant of S. typhimurium modulated the subsequent response to parental wild-type challenge and reduced carrier populations of wild-type S. typhimurium in infected swine.

Animals↗

PubMed1993

Identification and partial characterization of a group of weakly beta-hemolytic intestinal spirochetes of swine distinct from Serpulina innocens isolate B256.

Comparative analyses of a group of 16 weakly beta-hemolytic spirochetes isolated from feces and mucosal scrapings of intestines of swine in the midwestern United States, and eastern Canada revealed the existence of a phenotypically and genotypically related group of 7 isolates. Although isolates in this group differed from all known reference isolates of intestinal spirochetes of swine, partial similarity was detected with S. joneseae isolate 16, a newly identified weakly beta-hemolytic intestinal spirochete of human beings. In addition to producing weak beta-hemolysis on blood agar plates, S. innocens isolates B256 and 4/71, S. joneseae isolate 16, and the 16 field isolates lacked the characteristic ring phenomenon described for Serpulina hyodysenteriae, an enteropathogenic spirochete of swine. All but one of the field isolates of weakly beta-hemolytic intestinal spirochetes gave negative results for indole production. The same isolates yielded variable results for alpha-galactosidase production. By transmission electron microscopic examination of negatively-stained cross-sections of spirochetes, the isolates segregated into groups containing either 4 to 7 or 9 to 16 profiles of axial filaments per cell cross-section. Analyses of genomic DNA of selected isolates using whole-genome cross-hybridization revealed a single genetic type consisting of 7 field isolates of weakly beta-hemolytic intestinal spirochetes. The 7 field isolates were distinct from the reference isolates S. innocens isolates B256 and 4/71, S. hyodysenteriae isolates B78 and B204, and Treponema succinifaciens isolate 6091 based on the number of axial filaments per cell cross-section and lack of cross-hybridization signal. S. joneseae isolate 16, had the same number of axial filaments per cell cross-section and produced a weak hybridization signal with a representative isolate of the 7 weakly beta-hemolytic field isolates from swine. This report suggests the existence of a widely distributed group of closely related weakly beta-hemolytic intestinal spirochetes of swine with genotypic characteristics distinct from S. innocens isolate B256.

Animals↗

PubMed1992

A new balloon-expandable tantalum coil stent: angiographic patency and histologic findings in an atherogenic swine model.

The angiographic patency and histologic characteristics of a new balloon-expandable tantalum stent were studied after implantation intervals ranging from 1 to 32 weeks in atherogenic miniature swine peripheral and coronary arteries. Stents were placed in 34 arteries (10 coronary and 24 iliac arteries) in a total of 13 swine. Two swine died within 24 h of stent implantation. Follow-up angiography was performed before death was induced in 11 swine (8 coronary and 19 iliac arteries) and revealed 100% patency without evidence of lumen stenosis, thrombosis or migration of the stents. The neointimal thickening was maximal at 4 weeks after stent implantation and was at its minimum at 32 weeks after implantation with reendothelialization of the stents generally complete at that time. An advantage of this balloon-expandable device is its inherent longitudinal flexibility. The coil configuration allowed the nondeployed stent to negotiate acute bends in coronary arteries to reach the site of implantation and also allowed the deployed stent to conform to the natural contour of tortuous coronary arteries. The tantalum device was remarkable for its radiographic visibility, which greatly aided its placement under fluoroscopic guidance. This study demonstrates this stent's ease of implantation, excellent patency rate and absence of restenosis due to neointimal proliferation for up to 8 months in this atherogenic swine model.

Angioplasty, Balloon, Coronary↗

PubMed2004

Development of nested RT-PCR for the detection of swine hepatitis E virus in formalin-fixed, paraffin-embedded tissues and comparison with in situ hybridization.

Swine hepatitis E virus (HEV) was detected in formalin-fixed, paraffin-embedded tissues from naturally infected pigs by nested reverse transcription-polymerase chain reaction (RT-PCR). The results for seminested RT-PCR were compared with those determined by in situ hybridization. The results obtained show that use of xylene deparaffinization, digestion with proteinase K followed by nested RT-PCR is a reliable detection method. Swine HEV nucleic acid was detected in formalin-fixed, paraffin-embedded hepatic tissues from 40 pigs. Distinct positive signals for swine HEV were obtained in the same hepatic tissues by in situ hybridization. Swine HEV nucleic acid was localized to the cytoplasm of hepatocytes and had a granular staining pattern. The rate of agreement between nested RT-PCR and in situ hybridization for the detection of swine HEV in formalin-fixed, paraffin-embedded hepatic tissues was 100%.

Animals↗

PubMed2005

Molecular cloning, expression and functional characterization of miniature swine CD86.

CD86 is one of the key molecules involved in the co-stimulation of T cells. The complete cDNA encoding CD86 molecule of miniature swine was cloned and analyzed. A comparison of two CD86 amino acid sequences of miniature swine and domestic swine showed only three amino acid differences suggesting that it is unlikely to affect the major structural features of the miniature swine CD86 (msCD86). In the expression study, constitutive expression of CD86 mRNA was detected in various tissues, and the aberrant expression of the transcriptional variant (putative soluble form) was noted. The cDNA and amino acid sequences for this variant were determined and compared with those for the human soluble CD86, which was previously reported to co-stimulate the T cells. Interestingly, an alignment of the two sequences revealed that 51 amino acids corresponding to the sequence for the boundary of the extracellular and intracellular domains including the transmembrane domain are deleted at almost an identical location within the full form of CD86 from both species. This suggests the possibility of a co-stimulatory function of the putative soluble msCD86. In order to determine if the cloned msCD86 molecules has co-stimulatory activity, the proliferative responses of the human CD4(+) T cells to the msCD86-transfected COS cells were measured in the presence of Con A. The results revealed that CD86/COS, but not the mock/COS, efficiently co-stimulated the proliferation of the Con A-stimulated CD4(+) T cells and this co-stimulatory effect was blocked by CTLA4-Ig. The structural and functional information on the miniature swine CD86 from this study will enable a further genetic manipulation of CD86 as a therapeutic strategy for controlling the xenogeneic T cell immune responses mediated by the CD86-CD28 signal pathway.

Amino Acid Sequence↗

PubMed2006

Clinical signs for identification of neurocysticercosis in swine naturally infected with Taenia solium.

Taenia solium infection is a zoonotic disease and swine is the natural intermediate host. Till date no literatures have described clinical signs in swine indicative of brain involvement by cysticerci. In the present study we describe such clinical signs of porcine neurocysticercosis (NCC). These signs were excessive salivation, excessive blinking and tearing, and subconjunctival nodule. A total of 30 swine (18 with 2 or all 3 clinical signs and 12 without any sign) underwent magnetic resonance imaging (MRI). All 18 swine with above signs had NCC on MRI along with variable involvement of other organs that were subsequently confirmed by ex vivo MRI, necropsy and histopathology, while none of the 12 animals without any sign had NCC. As development of a porcine NCC model has proved difficult, we propose that naturally infected swine can be identified on the basis of these clinical signs and thus used as a model for further research on NCC.

Animals↗

PubMed2004

Molecular cloning of the swine IL-4 receptor alpha and IL-13 receptor 1-chains: effects of experimental Toxoplasma gondii, Ascaris suum and Trichuris suis infections on tissue mRNA levels.

IL-4 and IL-13 are multi-functional cytokines with overlapping roles in the host defense against infection. Equally important in the regulation of IL-4 and IL-13 are their associated receptors. Though, their functional receptor complexes and signaling pathways are intricate and in some cases, share common elements, the specificity of the responses, nonetheless, resides in the structure and binding of the alpha-chain components. This report presents the cloning of the swine receptors IL-4Ralpha and IL-13Ralpha1 and the effects of parasite infection on their transcription. Pairwise alignment of predicted amino acid sequences indicates that the swine IL-13Ralpha1 is 86, 83, and 72% similar to canine, human and mouse sequences, respectively. Amino acid sequence conservation is appreciably lower between the swine IL-4Ralpha sequence and those from equine (72%), human (66%), and mouse (49%); however, noteworthy similarities were observed in their overall predicted secondary structures predominantly among the swine, equine, and human homologues. Relative levels of receptor mRNA in tissues from swine experimentally infected with the protozoan, Toxoplasma gondii (T. gondii) or the nematodes Ascaris suum (A. suum) or Trichuris suis (T. suis), which are known to induce Th1 or Th2 host responses, respectively, were measured by real-time PCR. Results indicated that within 14 days following infection, overall mRNA levels for IL-4Ralpha and IL-13Ralpha1 were elevated in T. gondii-infected animals and reduced in A. suum-infected animals. Levels of swIL-4Ralpha and swIL-13Ralpha1 mRNA in T. suis-infected animals varied coincidentally with the course of the infection and the location of the analyzed tissue.

Amino Acid Sequence↗

PubMed2004

Longitudinal study of Salmonella species in 90 Alberta swine finishing farms.

The aim of this study was to determine the farm prevalence of Salmonella in 90 Alberta finishing swine farms over a 5-month period, to evaluate Salmonella distribution in the farm environment and to describe Salmonella serovar diversity on these farms. Ten veterinary practitioners selected 90 Alberta swine farms based on an annual production of > or =2000 market pigs per farm and the willingness of the producers to participate in the study. Between May and September 2000, twenty samples were collected from finishing swine and the environment of each farm. The annual production of selected farms represented approximately 25% of the market swine production in Alberta. Participating farms were geographically representative of major swine production areas in Alberta. Sixty (66.7%) farms had at least one Salmonella-positive sample, with confidence interval (CI) of 57.1-77.2%. Salmonella were detected in 14.3% of fecal and 20.1% of environmental samples. The number of Salmonella-positive samples per farm ranged from 1 to 19. Among environmental samples, Salmonella were most frequently recovered from boots (38.6%) and the main drain (31.8%). Twenty-two serovars were detected on the 60 Salmonella-positive farms. Serovars Typhimurium (78 isolates), Derby (71 isolates) and Infantis (47 isolates) were the most common. A single serovar was detected on 58 farms, while 2, 3 and >3 serovars were detected on 15, 10 and 7 farms, respectively. The Salmonella farm status changed frequently over the 5-month period indicating the dynamic nature of Salmonella infections on these farms.

Alberta↗

PubMed2004

Multiple lineages of antigenically and genetically diverse influenza A virus co-circulate in the United States swine population.

Before the isolation of H3N2 viruses in 1998, swine influenza in the United States was an endemic disease caused exclusively by classical-swine H1N1 viruses. In this study we determined the antigenic and phylogenetic composition of a selection of currently circulating strains and revealed that, in contrast to the situation pre-1998, the swine population in the United States is now a dynamic viral reservoir containing multiple viral lineages. H3N2 viruses still circulate and representatives of each of two previously identified phylogenetic groups were isolated. H1N1 and H1N2 viruses were also identified. In addition to the genotypic diversity present, there was also considerable antigenic diversity seen. At least three antigenic profiles of H1 viruses were noted and all of the recent H3N2 viruses reacted poorly, if at all, to the index A/swine/Texas/4199-2/98 H3N2 antiserum in hemagglutination inhibition assays. The influenza reservoir in the United States swine population has thus gone from a stable single viral lineage to one where genetically and antigenically heterogenic viruses co-circulate. The growing complexity of influenza at this animal-human interface and the presence of viruses with a seemingly high affinity for reassortment makes the United States swine population an increasingly important reservoir of viruses with human pandemic potential.

Animals↗

PubMed1996

Immunophenotypic characterization of tumor infiltrating lymphocytes and peripheral blood lymphocytes isolated from melanomatous and non-melanomatous Sinclair miniature swine.

Selectively-bred Sinclair miniature swine exhibit a high incidence of congenital malignant melanoma which either proves fatal (10-15% of tumor-bearing piglets) or spontaneously regresses with a biphasic immunological phenomenon (85-90%) and no recurrence of malignancy. Mononuclear leukocytes were isolated from cutaneous melanomas and peripheral blood specimens collected from melanomatous (tumor-bearing) Sinclair swine during second-phase regression, and from peripheral blood specimens collected from non-melanomatous (tumor-free) Sinclair swine and control Hanford swine. Leukocyte identities were determined with single- and dual-parameter indirect immunofluorescence assays via flow cytometry. Assays for the specific surface antigens CD45, CD2, CD4, CD8, CD1, MHC class II, and N1 were employed to develop immunophenotypic profiles within the gated lymphocyte clusters from each TIL and PBL suspension. Significantly more CD8+ T-lymphocytes were identified in TIL suspensions than in peripheral blood leukocyte (PBL) suspensions (P < and = 0.05), regardless of breed or tumor status. Conversely, PBL suspensions contained significantly higher percentages of CD4+ T-lymphocytes than the levels found in TIL suspensions (P < and = 0.05). Virtually all TIL were MHC class II+, whereas the percentages of PBL expressing this antigen were markedly lower (P < and = 0.05). The percentages of T-lymphocytes co-expressing CD4 and CD8, a normal subset unique to swine, were generally consistent in all TIL and PBL suspensions examined. The results of this study have firmly established the immunophenotypic identities of cells associated with the second-phase regression phenomenon of this melanoma and have identified specific variations in the leukocyte profiles of the respective TIL and PBL suspensions.

Animals↗

PubMed1998

Construction of internal cDNA competitors for measuring IL-10 and IL-12 cytokine gene expression in swine.

A competitive PCR assay (cPCR) was used to quantify swine cytokine responses to parasite infection. Internal standards (deleted cDNA competitor molecules [DcDNA mimics]) were produced and tested for swine interleukin-12 (IL-12), interleukin-10 (IL-10) and hypoxanthine phosphoribosyltransferase (HPRT) from PCR generated cDNA cloned in plasmid vectors. Deletion clones for the cDNA competitor molecules (DcDNA mimics) were generated for IL-10, IL-12 and HPRT by PCR in a single step and verified by (1) amplification of the expected smaller PCR product with the original primers, (2) appropriate fragment size released by restriction digestion of the deleted clone, and (3) correct sequence of the new DcDNA insert. DcDNA mimics were used to quantitate cytokine gene mRNA production during experimental and natural infections of swine with the gastrointestinal nematode parasite Trichuris suis. Mesenteric lymph node cells were collected from control and infected pigs at the time of maximal pathogenicity (35 days after infection) and snap frozen. After RNA extraction, samples were reverse transcribed (RT) to cDNA. cPCR was performed using the housekeeping gene HPRT DcDNA mimic and HPRT specific primers to insure RNA integrity and concentration. Cytokine cDNA content in these samples was then quantitated using cytokine mimics and gene specific primers. IL-10 gene expression in MLN draining the colon of pigs experimentally infected with T. suis increased 10-20 fold at day 35 compared to control pigs. IL-12 gene expression was not detectable in MLN of these pigs, but was detectable in MLN of pigs exposed naturally to T. suis on a contaminated dirt lot that also exhibited signs of secondary bacterial invasion. Swine IL-10 and IL-12 gene expression can be quantitated in local mesenteric tissues. This cPCR assay will enable scientists to quantitate cytokine gene expression in swine and determine the nature of immune responses to important infectious diseases.

Animals↗