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A safe, efficacious, and clinically applicable immunosuppressive regimen is necessary for islet xenotransplantation to become a viable treatment option for diabetes. We performed intraportal transplants of wild‐type adult porcine islets in 25 streptozotocin‐diabetic cynomolgus monkeys. Islet engraftment was good in 21, partial in 3, and poor in 1 recipient. Median xenograft survival was 25 days with rapamycin and CTLA4Ig immunosuppression. Adding basiliximab induction and maintenance tacrolimus to the base regimen significantly extended median graft survival to 147 days (p < .0001), with three animals maintaining insulin‐free xenograft survival for 265, 282, and 288 days. We demonstrate that this regimen suppresses non‐Gal anti‐pig antibody responses, circulating effector memory T cell expansion, effector function, and infiltration of the graft. However, a chronic systemic inflammatory state manifested in the majority of recipients with long‐term graft survival indicated by increased neutrophil to lymphocyte ratio, IL‐6, MCP‐1, CD40, and CRP expression. This suggests that this immunosuppression regimen fails to regulate innate immunity and resulting inflammation is significantly associated with increased incidence and severity of adverse events making this regimen unacceptable for translation. Additional studies are needed to optimize a maintenance regimen for regulating the innate inflammatory response. A clinically available immunosuppressive regimen prevents rejection of wild‐type porcine islets in cynomolgus macaques but fails to control xenogeneic systemic inflammation, previously unrecognized in cell transplantation, thereby precluding its direct applicability.

A safe, efficacious, and clinically applicable immunosuppressive regimen is necessary for islet xenotransplantation to become a viable treatment option for diabetes. We performed intraportal transplants of wild-type adult porcine islets in 25 streptozotocin-diabetic cynomolgus monkeys. Islet engraftment was good in 21, partial in 3, and poor in 1 recipient. Median xenograft survival was 25 days with rapamycin and CTLA4Ig immunosuppression. Adding basiliximab induction and maintenance tacrolimus to the base regimen significantly extended median graft survival to 147 days (p< .0001), with three animals maintaining insulin-free xenograft survival for 265, 282, and 288 days. We demonstrate that this regimen suppresses non-Gal anti-pig antibody responses, circulating effector memory T cell expansion, effector function, and infiltration of the graft. However, a chronic systemic inflammatory state manifested in the majority of recipients with long-term graft survival indicated by increased neutrophil to lymphocyte ratio, IL-6, MCP-1, CD40, and CRP expression. This suggests that this immunosuppression regimen fails to regulate innate immunity and resulting inflammation is significantly associated with increased incidence and severity of adverse events making this regimen unacceptable for translation. Additional studies are needed to optimize a maintenance regimen for regulating the innate inflammatory response.

Numerous interspecies disease transmission events, Ebola virus being a recent and cogent example, highlight the complex interactions between human, animal, and environmental health and the importance of addressing medicine and health in a comprehensive scientific manner. The diversity of information gained from the natural, social, behavioral, and systems sciences is critical to developing and sustainably promoting integrated health approaches that can be implemented at the local, national, and international levels to meet grand challenges. The Concept of One Medicine One Science (COMOS) as outlined herein describes the interplay between scientific knowledge that underpins health and medicine and efforts toward stabilizing local systems using 2 linked case studies: the food system and emerging infectious disease. Forums such as the International Conference of One Medicine One Science (iCO-MOS), where science and policy can be debated together, missing pieces identified, and science-based collaborations formed among industry, governmental, and non-governmental policy makers and funders, is an essential step in addressing global health. The expertise of multiple disciplines and research foci to support policy development is critical to the implementation of one health and the successful achievement of global health security goals.

Abstract: The incidence of calcium oxalate (CaOx) urolithiasis in dogs has increased steadily over the last two decades. A potential mechanism to minimize CaOx urolithiasis is to reduce enteric absorption of dietary oxalate by oxalate-metabolizing enteric bacteria. Enteric colonization of Oxalobacter formigenes, an anaerobe which exclusively relies on oxalate metabolism for energy, is correlated with absence of hyperoxaluria or CaOx urolithiasis or both in humans and laboratory animals. We thus hypothesized that decreased enteric colonization of O. formigenes is a risk factor for CaOx urolithiasis in dogs. Fecal samples from dogs with CaOx uroliths, clinically healthy, age-, breed- and gender-matched dogs, and healthy non-stone forming breed dogs were screened for the presence of O. formigenes by quantitative PCR to detect the oxalyl CoA decarboxylase (oxc) gene, and by oxalate degrading biochemical activity in fecal cultures. Prevalence of O. formigenes in dogs with CaOx uroliths was 25%, compared to 50% in clinically healthy, age-, breed- and gender-matched dogs, and 75% in healthy non-stone forming breeds. The presence of oxc genes of O. formigenes was significantly higher in healthy non-stone forming breed dogs than in the dogs with CaOx stones. Further, dogs with calcium oxalate stones and the stone-forming breed-matched controls showed comparable levels of biochemical oxalate degrading activity. We conclude that the absence of enteric colonization of O. formigenes is a risk factor for CaOx urolithiasis.

ABSTRACTPorcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) are major contributors to the porcine respiratory disease complex (PRDC). Routine serological diagnosis and surveillance play an important role in the prevention of PRDC, as it is a leading cause of economic losses to the swine industry. We herein describe an advanced microsphere-based immunoassay that permits the simultaneous detection of antibodies to PCV2 and PRRSV, thereby reducing the time and effort involved in testing. Recombinant PRRSV nucleoprotein antigen and the PCV2 capsid antigen were coupled to fluorophore-dyed beads with distinct spectral addresses. Weekly serum samples from 72 pigs that were experimentally exposed to either PCV2, PRRSV, or both PCV2 and PRRSV were used to validate the microbead assay (MBA) in comparison with the “gold standard” enzyme-linked immunosorbent assays. The kinetics of the PCV2- and PRRSV-specific antibody responses measured by the microbead assay were comparable to those of the standard assays; Spearman's rank correlations were 0.72 (P< 0.001) for PRRSV and 0.80 (P< 0.001) for PCV2. Diagnostic sensitivity and specificity were determined using field sera whose positive or negative status was determined by the standard tests. The diagnostic sensitivity and specificity were both 98% for PCV2 and were 91% and 93%, respectively, for PRRSV (kappa coefficients, 0.85 and 0.67 for PCV2 and PRRSV, respectively). Multiplexing did not interfere with assay performance or diagnostic sensitivity. Therefore, the described study demonstrates proof of concept for the development of more versatile and economical microbead array-based multiplex serological test panels for veterinary use.

A blinded interlaboratory assessment of the diagnostic agreement and accuracy of serologic tests for routine detection of antibodies against Porcine circovirus-2(PCV-2), including indirect fluorescent antibody tests (IFATs) and enzyme-linked immunosorbent assays (ELISAs) was conducted in 7 North American laboratories. Serum samples were collected weekly, on trial days 0, 7, 14, 21, 28, 35, 42, and 49, from the following groups of animals: 1) negative controls (n= 7), 2) PCV-2a (n= 8), 3) PCV-2b (n= 8), 4) PCV-1 (n= 8), 5) PCV-2 vaccine A (n= 8; Ingelvac® CircoFLEX™), 6) PCV-2 vaccine B (n= 8; Circumvent® PCV2), and 7) PCV-2 vaccine C (n= 8; Suvaxyn® PCV2 One Dose). Results from each laboratory were analyzed by kappa and receiver operating characteristic (ROC) analysis. Kappa analysis indicated that, by trial day 49, IFATs had almost perfect agreement, in-house ELISAs had fair to almost perfect agreement, and commercially available anti–PCV-2 immunoglobulin G ELISAs (I or S) had moderate to substantial agreement. From trial days 14–49, the area under the ROC curve for the 2 laboratories that offered IFATs, the 4 laboratories that offered in-house ELISAs, and the 3 laboratories that used commercially available ELISAs ranged from 0.94 to 1.00, 0.72 to 1.00, and 0.95 to 1.00, respectively. However, test sensitivities varied based on laboratory-specific cutoffs that were used to dichotomize test results.

ABSTRACTTo further characterize the humoral immune response of pigs to porcine reproductive and respiratory syndrome virus (PRRSV), direct enzyme-linked immunosorbent assays (ELISA) were used to study the kinetics of antibody responses directed against PRRSV nonstructural proteins in pigs experimentally exposed to the virus. The highest immunoreactivities were against nsp1, nsp2, and nsp7. Using the recombinant nsp7 as an antigen, we validated a dual ELISA for the simultaneous detection and differentiation of serum antibodies against type I and type II PRRSV. Receiver operating characteristic analysis based on 1,334 known-positive and 1,357 known-negative samples showed good specificity (98.3% to type I and 99.3% to type II) and sensitivity (97.4% for type I and 99.8% for type II). To differentiate type I and type II PRRSV, 470 sera originating from experimentally inoculated pigs were tested, and positive sera were correctly differentiated in 469 of 470 samples. The capability of the nsp7 dual ELISA to detect serum antibody responses from pigs infected with various genetically different field strains was determined. The nsp7 dual ELISA possessed 97.6% agreement with the Idexx HerdChek PRRS 2XR ELISA. In further testing of Idexx ELISA suspected false-positive samples, the nsp7 dual ELISA resolved 98% of the samples as negative. Taken together, these results indicate that the nsp7 dual ELISA can be used as a differential test for PRRSV serology with high levels of sensitivity and specificity. This ELISA offers an additional tool for routine or follow-up diagnostics, as well as having substantial value in epidemiological surveys and outbreak investigations.

Porcine reproductive and respiratory syndrome virus(PRRSV) is an important pathogen of swine. The objective of the current study is to investigate the feasibility of using reverse transcription loop-mediated isothermal amplification (RT-LAMP) for the detection of PRRSV. The RT-LAMP is a recently described DNA amplification technique reported to be simple, inexpensive, fast, and accurate. The RT-LAMP reaction was set up using 2 sets of primers that were designed to detect North American and European strains of PRRSV and performed successfully in a simple heat block. The specificity of the amplified product was demonstrated by restriction analysis. The RT-LAMP was able to detect 5 different PRRSV isolates. However, the limit of detection ranged between 102and 10450% tissue culture infective dose/ml. The RT-LAMP was further evaluated using serum samples from animals of known infection status. The ability of RT-LAMP to detect PRRSV in serum from acutely infected animals was evaluated with 114 serum samples from 18 experimentally inoculated boars. Forty-nine of these samples tested positive by RT-LAMP, while 94 were positive by reverse transcription polymerase chain reaction (RT-PCR). The diagnostic specificity, evaluated with 100 known negative serum samples, was estimated as 99%. The feasibility of RT-LAMP to detect PRRSV was demonstrated in the current study. The RT-LAMP reaction could be performed in just 1 hr with a simple and inexpensive heat block. However, the sensitivity of this technique was significantly lower than that of RT-PCR.

The enteric immune system of swine protects against infectious and noninfectious environmental insults and discriminates ingested nutrients, food, and commensal microflora from pathogenic agents. The molecular and cellular elements of the immune system have been selected over evolutionary time in response to the specific environment of pigs. Thus, models of immune function based on mouse and human need to be applied cautiously in the pig. To better understand how the mucosal immune system of the small intestine accomplishes the conflicting functions of food tolerance and immunity to enteric infection, we used a genomic approach to profile gene expression in the Peyer's patch. More than 40% of mRNA enriched by differential subtraction for Peyer's patch-specific expressed sequences represented genes of unknown function or had no match in GenBank. Microarray analysis and radiation hybrid mapping validated their porcine origin and provided additional insights into putative functions. The abundance of expressed genes of unknown function indicates that a substantial fraction of the immunological and physiological processes of the Peyer's patch remains to be discovered. It further suggests that swine have evolved specialized biochemical and immunological processes in the small intestine. Further elucidation of these processes are expected to provide novel insights into swine enteric mucosal immune function.

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be one of the most important diseases facing swine industry today. Following PRRSV infection pigs develop both humoral and cell-mediated responses following PRRSV exposure; however, the relative importance in protection and clearance of the virus is not yet completely understood. Swine contain a large percentage of γγδδ T-lymphocytes in peripheral circulation capable of responding to various pathogens in both an innate and specific immune response. The objectives of this study were to determine whether γγδδ lymphocytes functionally respond to PRRSV upon initial exposure and re-exposure. Four month old PRRSV free gilts were intranasally inoculated with a field isolate MN-30100 then assessed at various time points post infection. On day 120, pigs were re-exposed with MN-30100 PRRSV strain and subsequently were bled on days 0, 7, and 14 post re-exposure. Lymphocyte subpopulations, antigen specific proliferation, and IFN-γγ production were evaluated throughout the study. Circulating γγδδ lymphocytes in PRRSV exposed animals expanded between days 14 to 70 (d14––d70, p= 0.016); following antigen stimulation, γγδδ lymphocyte proliferated by day 14 (d0––d14, p= 0.001) continuing through day 60. γγδδ lymphocytes produced IFN-γγ by day 14 pi continuing through day 50 (d0––d50, p= 0.004). Following re-exposure both γγδδ+and CD4+lymphocytes increased in IFN-γγ production. These results are not fully conclusive on the role of γγδδ lymphocytes against PRRSV; the data indicate that γγδδ lymphocytes specifically respond to PRRSV.

ABSTRACTPorcine reproductive and respiratory syndrome (PRRS) continues to be one of the most significant diseases of swine. IDEXX HerdChek PRRS, a commercially available enzyme-linked immunosorbent assay (ELISA), has become the industry standard for the detection of antibodies against PRRS virus (PRRSV). The need to accurately determine the PRRSV serostatus of herds and individual animals has prompted the development of several follow-up assay methods. A highly specific and repeatable blocking ELISA (bELISA) was developed on the basis of the use of an expressed PRRSV nucleocapsid (N) protein as the antigen and a biotinylated monoclonal antibody. Validation of the bELISA used sera from 316 animals experimentally and naturally infected with North American PRRSV and sera from 370 uninfected animals. Receiver operating characteristic analysis of the data calculated a diagnostic sensitivity of 97.8% and a diagnostic specificity of 100%. The between-run coefficient of variation of an internal quality control serum was 4.24%. The bELISA was able to detect seroconversion as well as the IDEXX ELISA and the indirect fluorescent antibody (IFA) assay; kappa values were 0.94 and 0.96, respectively. A collection of 133 serum samples with unexpected positive IDEXX ELISA results was obtained from 4,038 diagnostic samples submitted from farms from which PRRS-negative results were expected. The bELISA identified 97% of the samples as PRRS seronegative, while the IFA identified 100% as seronegative. The anticipated use of the bELISA is as a follow-up test to the IDEXX ELISA for determining the PRRSV serostatus of individual animals with unexpected positive test results from swine herds from which negative results are expected.

The immune response of swine to vaccination with a live, attenuated PRRSV was assessed in the presence and absence of cytokine adjuvants or cholera toxin (CT) to address the hypothesis that adjuvant danger signals, that is, inflammatory cytokines and bacterial extoxin, stimulate a more robust immune response. Animals received four injections of recombinant porcine IL-1 and IL-6, IL-12 alone, or CT alone within 1 week of intramuscular administration of a vaccine strain of PRRSV, Ingelvac MLV. Serological and cell-mediated responses were monitored for 42 days after vaccination and for a further 10 days after challenge with the virulent VR2332 strain of PRRSV. First, the principal observation was an enhancing effect of IL-12 on the interferon γ response to PRRSV, with a more rapid and heightened PRRSV-specific interferon γ ELISPOT response in peripheral blood mononuclear cells. The more rapid and robust development of a cell-mediated immune response, as determined by this assay, suggests that IL-12 may influence the induction of antigen-specific T cell responses. Second, animals that received CT adjuvant displayed a more robust antibody response to GP5, the major envelope glycoprotein. Anti-GP5 titers peaked at 21 days after vaccination at more than twice the level of any other treatment, for which the peak response was at 28 days. Third, there was no evidence of PRRSV immunosuppression of immunity to unrelated antigens, including circovirus. CT is a potent mucosal adjuvant, particularly for antibody responses. It acts in part through the production of IL-1 in macrophages, but its effect was not replaced by combination treatment with IL-1 and IL-6. In sum, the results suggest that cytokine adjuvants, particularly IL-12, and CT have the potential to enhance immune responses to live viral vaccines.

The immunology of porcine reproductive and respiratory syndrome virus (PRRS) begins with an initial encounter of PRRSV with the pig. Regardless of the route of entry of PRRSV—via inhalation, intramuscular vaccination, insemination, or other routes—productive infection occurs predominately in alveolar macrophages of the lung. Thus, innate responses of the lung and the alveolar macrophage comprise the initial defense against PRRSV. The virus appears not to elicit innate interferon and cytokine responses characteristic of other strongly immunogenic viral pathogens, and its effects are consistent with induction of a weak adaptive immune response. Humoral and cell-mediated immunity is induced in due course, and results in clearance of virus from the circulation but not from lymphoid tissues, where the infection becomes persistent. Subsequent reexposure to PRRSV elicits an anamnestic response that is partially to completely protective. Within this unconventional picture of anti-PRRSV immunity lie a variety of unresolved issues, including the nature of protective immunity within individual pigs and among pigs in commercial populations, the efficacy of protective immunity against genetically different PRRSV isolates, the effects of developmental age, sex, genetics, and other host factors on the immune response to PRRSV, and the possible suppression of host immunity to other pathogens.

Hormones influence uterine contractility through their effects on intracellular calcium. The regulation of intracellular calcium in uterine smooth muscle is achieved by several mechanisms and includes mobilization from intracellular stores by inositol 1,4,5-trisphosphate and ryanodine-sensitive channels. Cyclic ADP-ribose (cADPR), a metabolite of NAD+, is known to mediate calcium release through ryanodine receptor channels. A cell surface glycoprotein, CD38, catalyzes the synthesis and breakdown of cADPR and thus possesses bifunctional enzymatic activity. The regulation of cADPR synthesis by ADP-ribosyl cyclase (cyclase) or degradation by cADP-ribose hydrolase (hydrolase) by hormones in the myometrium is poorly understood. We investigated the effects of estradiol-17β on CD38 expression and the synthesis and degradation of cADPR in myometrial smooth muscle obtained from ovariectomized rats. CD38 expression was studied by reverse transcription polymerase chain reaction and Western blot analyses. In uterine microsomal fractions, cyclase and hydrolase activities were measured using nicotinamide guanine dinucleotide and [32P]cADPR as substrates, respectively. Microsomal proteins subfractionated by SDS-PAGE and gel filtration were used to determine the fractions containing cyclase and hydrolase activities. The results demonstrate that cyclase and hydrolase activities are associated with a single protein fraction, similar to CD38 in uteri from both ovariectomized and estradiol-treated rats, and estradiol-17β causes 1) increased CD38 mRNA and protein expression and 2) significantly enhanced cyclase but not hydrolase activity. The differential regulation of CD38 by estradiol-17β, resulting in increased cADPR synthesis, would have profound effects on calcium regulation and myometrial contractility.

ABSTRACTRecent advances toward using pig tissues in human transplantation have made it necessary to determine the risk of transmitting zoonotic viruses from pigs to humans or vice versa. We investigated the suitability of the porcine encephalomyocarditis virus (EMCV) model for such studies by determining its ability to persist in pigs, escape detection by routine serological methods, and infect human cells. Intraperitoneal inoculation of 5-week-old pigs with EMCV-30, a strain isolated from commercial pigs, resulted in acute cellular degeneration, infiltration of lymphocytes, and apoptosis in myocardium in 13 of 15 (86.7%) pigs during the acute phase of disease (3 to 21 days postinfection), followed by less-severe lymphocytic infiltration and apoptosis in 5 of 10 (50%) pigs during the chronic phase of the disease (day 45 to 90 postinfection). In the brain, lymphocytic infiltration, neuronal degeneration, and gliosis were observed in 26 to 33% of pigs in the acute phase of disease whereas perivascular cuffing was the predominant feature during chronic disease. EMCV antigens and RNA were demonstrated in the myocardium and brain during the chronic phase of disease. Analysis of 100 commercial pigs that were negative for EMCV antibodies identified two pig hearts positive for EMCV RNA. Porcine EMCV productively infected primary human cardiomyocytes as demonstrated by immunostaining using a monoclonal antibody specific for EMCV RNA polymerase, which is expressed only in productively infected cells, and by a one-step growth curve that showed production of 100 to 1,000 PFU of virus per cell within 6 h. The findings that porcine EMCV can persist in pig myocardium and can infect human myocardial cells make it an important infectious agent to screen for in pig-to-human cardiac transplants and a good model for xenozoonosis.

Porcine reproductive and respiratory syndrome virus (PRRSV) was shown to produce atypical subgenomic RNAs that contain open reading frame 1a nucleotides and are present under a wide variety of culture conditions, including high and low multiplicities of infection, in simian and porcine host cells, and during infection with cell-adapted and wild-type PRRSV strains. Sequence analysis demonstrated that they are heterogeneous in 5′–3′ junction sequence and size and may code for different predicted fusion proteins. This is the first report of these novel RNAs in arteriviruses and we have termed them heteroclite (meaning “deviating from common forms or rules”) subgenomic RNAs. The unique properties of these subgenomic RNAs include (a) apparent association with normal virus infection and stability during serial passage, (b) packaging of heteroclite RNAs into virus-like particles, (c) short, heterogeneous sequences which may mediate the generation of these RNAs, (d) a primary structure which consists of the two genomic termini with one large internal deletion, and (e) little apparent interference with parental virus replication. These subgenomic RNAs may be critical to, or a necessary side product of, viral replication. The expression of these novel RNA species support the template-switching model of similarity-assisted RNA recombination. In summary, PRRSV readily undergoes nonhomologous RNA recombination to generate heteroclite subgenomic RNAs.

ABSTRACTRespiratory infection by Actinobacillus pleuropneumoniaecauses a highly pathogenic necrotizing pleuropneumonia with severe edema, hemorrhage and fever. Acute infection is characterized by expression of inflammatory cytokines, including interleukin-1 (IL-1), IL-6 and IL-8. To determine if high level production of inflammatory cytokines contributed to disease pathogenesis, we investigated if inhibiting macrophage activation with adenovirus type 5-expressed IL-10 (Ad-5/IL-10) reduced the severity of acute disease. Porcine tracheal epithelial cells infected with Ad-5/IL-10 produced bioactive human IL-10. When pigs were intratracheally infected with A. pleuropneumoniae, pigs pretreated with Ad-5/IL-10 showed a significant reduction in the amount of lung damage when compared to adenovirus type 5-expressing β-galactosidase (Ad-5/β-Gal)-treated and untreated pigs. In addition, serum zinc levels were unchanged, the lung weight/body weight ratio (an indicator of vascular leakage) was significantly reduced, and lung pathology scores were reduced. Myeloperoxidase activity in lung lavage fluid samples, an indicator of neutrophil invasion, was decreased to levels similar to that seen in pigs not infected withA. pleuropneumoniae. Reduction in inflammatory cytokine levels in lung lavage fluid samples correlated with the clinical observations in that pigs pretreated with Ad-5/IL-10 showed a corresponding reduction of IL-1 and tumor necrosis factor (TNF) compared with untreated and Ad-5/β-Gal-treated pigs. IL-6 levels were unaffected by pretreatment with Ad-5/IL-10, consistent with observations that IL-6 was not derived from alveolar macrophages. Since inflammatory cytokines are expressed at high levels in acute bacterial pleuropneumonia, these results indicate that macrophage activation, involving overproduction of IL-1 and TNF, is a prime factor in infection-related cases of massive lung injury.

ABSTRACTPorcine reproductive and respiratory syndrome virus (PRRSV) is a recently described arterivirus responsible for disease in swine worldwide. Comparative sequence analysis of 3'-terminal structural genes of the single-stranded RNA viral genome revealed the presence of two genotypic classes of PRRSV, represented by the prototype North American and European strains, VR-2332 and Lelystad virus (LV), respectively. To better understand the evolution and pathogenicity of PRRSV, we obtained the 12,066-base 5'-terminal nucleotide sequence of VR-2332, encoding the viral replication activities, and compared it to those of LV and other arteriviruses. VR-2332 and LV differ markedly in the 5' leader and sections of the open reading frame (ORF) 1a region. The ORF 1b sequence was nearly colinear but varied in similarity of proteins encoded in identified regions. Furthermore, molecular and biochemical analysis of subgenomic mRNA (sgmRNA) processing revealed extensive variation in the number of sgmRNAs which may be generated during infection and in the lengths of noncoding sequence between leader-body junctions and the translation-initiating codon AUG. In addition, VR-2332 and LV select different leader-body junction sites from a pool of similar candidate sites to produce sgmRNA 7, encoding the viral nucleocapsid protein. The presence of substantial variations across the entire genome and in sgmRNA processing indicates that PRRSV has evolved independently on separate continents. The near-simultaneous global emergence of a new swine disease caused by divergently evolved viruses suggests that changes in swine husbandry and management may have contributed to the emergence of PRRS.

Serologic studies have revealed strain variability between American and European isolates and among American isolates of porcine reproductive and respiratory syndrome virus (PRRSV). The objective of this study was to develop an assay for the routine diagnosis of PRRSV in field specimens using reverse transcription-polymerase chain reaction (RT-PCR) amplification of conserved genomic regions. Twenty-four field isolates of PRRSV from different regions of the USA were analyzed in the study. Six primer pairs from open reading frames (ORFs) 4, 6, and 7 of the American strain (ATCC VR-2332) and from ORF 1b of the Lelystad strain were used for the amplification of the viral genome by PCR. Amplification products of the expected sizes were obtained from all isolates by PCR amplification of ORF 7, the gene encoding the nucleocapsid protein. Oligonucleotide primers designed to amplify ORFs 4 and 6 detected 92% and 96% of the isolates, respectively, whereas primers for the amplification of ORF 1b detected 88% of all isolates. The specificity of the amplified products of ORF 7 from 7 field isolates and 2 reference strains was confirmed by chemiluminescent hybridization using an internal digoxigenin-labeled DNA probe. Sequence analysis of this region indicated variation in the nucleotide sequence of 2 isolates that did not hybridize with the internal probe. These results indicate that ORF 7 may serve as a potential target for the detection of PRRSV strains by RT-PCR and that genomic variability should be considered when nucleic acid hybridization is used to confirm the specificity of PCR amplification for diagnostic purposes.

ABSTRACTThe ability of innate immune cells to differentially respond to various bacterial components provides a mechanism by which the acquired immune response may be tailored to specific pathogens. The response of innate immune cells to bacterial components provides regulatory signals to cognate immune cells. These signals include secreted cytokines and costimulatory molecules, and to a large extent they determine the quantitative and qualitative nature of the immune response. In order to determine if innate immune cells can differentially respond to bacterial components, we compared the responses of macrophages to two bacterially derived molecules, cholera toxin (CT) and lipopolysaccharide (LPS). We found that CT and LPS differentially regulated the expression of interleukin-12 (IL-12) and CD80-CD86 but not that of IL-1β. LPS and CT each induced IL-1β expression in macrophages, while only LPS induced IL-12 and only CT induced CD80-CD86. These differences were markedly potentiated in gamma interferon (IFN-γ)-treated macrophages, in which LPS potently induced IL-12 and CD80-CD86 expression. In contrast, IFN-γ treatment had no effect on the expression of IL-1β. These results define a molecular basis for the differential pathogenicities of bacterial toxins and are relevant to the design of vaccine adjuvants able to selectively induce desired types of immunity.

Drug‐specific monoclonal antibodies and their antigen‐binding Fab fragments reverse acute desipramine toxicity in a rat experimental model by inducing a redistribution of drug from cardiac tissue into serum and extracellular fluid. In order to investigate the use of smaller recombinant antibody fragments such as single chain Fv (sFv) as an antidote, an efficient murine NS/0 myeloma expression system was developed. The variable light (VL) and variable heavy (VH) domains of a murine anti‐desipramine monoclonal antibody were cloned and sequenced. A 270 amino acid VH‐(Gly4Ser)3‐VLsFv was prepared by overlapping polymerase chain reaction (PCR) amplification of VHwith heavy chain leader peptide, VL, and the linker. This construct was subcloned into a mammalian expression vector which utilizes the SRα promoter, a hybrid promoter consisting of the SV40 early promoter with portions of the human T‐cell leukemia virus type I long terminal repeat and also containing theEscherichia colixanthine–guanine phosphoribo‐syltransferase gene for selection. NS/0 myeloma cells were transfected by electroporation. Stable recombinant NS/0 clones were screened for expression of sFv using reverse transcriptase‐PCR to detect mRNA and an enzyme‐linked immunosorbent assay (ELISA) to detect sFv. Secreted sFv from clones capable of growth to a cell density of 2–4 × 106viable cells/mL was purified in a single step using a desipramine affinity column resulting in 12–39 mg/L of purified sFv. Affinity‐purified sFv had comparable desipramine binding activity to Fab when evaluated by competitive ELISA.

Neutrophil‐induced alterations in feline erythrocytes were studied to better understand the pathogenesis of erythrocyte destruction associated with inflammatory diseases. As in previous studies, addition of superoxide dismutase/catalase to a coculture of erythrocytes and activated neutrophils attenuated neutrophil‐induced immunoglobulin G (IgG) binding. However, incubation of erythrocytes with hydrogen peroxide or neutrophil‐derived anuclear cytoplasts (neutroplasts) failed to induce IgG binding. Addition of phenylmethylsulfonyl fluoride, a serine protease inhibitor, to the erythrocyte‐neutrophil coculture attenuated IgG binding. These observations suggest that neutrophil‐derived serine protease activity is involved in IgG binding to erythrocytes. Further, incubation of erythrocytes with serine proteases, but not metalloproteases or sulfhydryl proteases, induced immunoglobulin binding. Freeze‐fracture replicas of the erythrocyte membrane failed to demonstrate clustering of band 3 protein, suggesting that spatial rearrangement of band 3 protein was not the cause of the IgG binding. Neutrophil‐induced IgG binding due to the combined action of proteases and oxidants may explain the accelerated destruction of erythrocytes in inflammatory diseases.

In vitro exposure of the synthetic opiate drug methadone allowed evaluation of putative immunomodulatory activities of swine peripheral blood mononuclear cells. Respiratory burst, an index of microbicidal activity, was suppressed by methadone in a dose‐dependent manner following exposure for 48 h. The suppression was blocked by the opiate antagonist naloxone. Another macrophage function phagosome‐lysosome fusion was impaired by exposure to methadone. A primary lymphocyte‐mediated function natural killer cell activity was also affected. In contrast, the macrophage function antibody‐mediated phagocytosis was not affected. Because the functions affected by methadone are critical to host defenses against pathogenic organisms, our findings suggest that opiate‐mediated immunomodulation merits further study. Moreover, our studies suggest that swine may provide an ideal model for the investigation of opiate‐mediated suppression of immune cell functions.

Neutrophil‐induced alterations in feline erythrocytes were investigated to elucidate the pathogenesis off the erythrocyte destruction associated with inflammatory disease. Incubation of erythrocytes with zymosan‐activated neutrophils, at target:effector concentrations of 50:1, resulted in immunoglobulin binding to the erythrocyte membrane and degradation of membrane proteins. Vitamin E and superoxide dismutase/catalase attenuated the immunoglobulin binding. Proteolysis was not prevented by addition of antioxidants or protease inhibitors to the erythrocyte:neutrophil coculture and was not reproduced with chemical oxidation or calcium loading of erythrocytes.

Opiates have potent antidiarrheal actions thatare mediated in part by delta-opioid receptors (DOR). Weexamined DOR localization within subregions of porcineileum, a tissue analogous to human small bowel. A partial cDNA sequence for porcine DOR wasobtained after reverse transcription-polymerase chainreaction cloning of forebrain RNA; it encoded the end oftransmembrane domain 1 through the beginning of transmembrane domain 7 and exhibited 93%nucleotide identity with human DOR. Positive signals forDOR mRNA were found in all subregions of the porcineileal wall. With an antiserum recognizing an N-terminal epitope in murine DOR, DOR-likeimmunoreactivity was localized in neurons withinmyenteric and submucous ganglia, longitudinal andcircular smooth muscle, and villous lamina propria. TheDOR agonist [D-Ser2, Leu5, Thr6]enkephalin(DSLET) attenuated circular smooth muscle contractionsin porcine ileum that were evoked by electricalstimulation of myenteric cholinergic neurons. Theseresults are consistent with previous reports of the DOR-mediatedneuromodulation that underlies the antipropulsive andantisecretory effects of opioids in the intestinaltract.

This study examines the achievement of autonomy by a sample of 30 midly learning handicapped and 30 nonhandicapped adolescents. From participant observation over the course of a school year, a detailed account was obtained about the life of each subject, both in the home and among friends outside the home. Findings suggest that autonomy is achieved through a gradual process of negotiation during the high school years. The mildly handicapped adolescents generally lag behind their regular education counterparts in breaking away from the family. While almost three-fourths of the nonhandicapped sample had made significant gains toward increased freedom, less than half of the mildly handicapped sample had shed close supervision by parents. However, most of the handicapped adolescents were satisfied with the limited freedom they had. The data suggest that autonomy should be understood as relative to the needs and abilities of each individual. Not all adolescents develop at the same pace; not all feel the same desire to assert their independence from their parents when they reach a particular age.

The function of intracellular transglutaminases remains to be clarified. In fibroblasts the links between the activity of this enzyme and receptor-mediated endocytosis are complex and open to interpretation. However, the issue cannot be firmly laid to rest until the structural specificity of the alkylamine inhibitors of endocytosis is explained. In macrophages, there is substantial evidence that the enzyme plays some role in receptor-mediated phagocytosis, but what this role is and how it might relate to endocytosis in other types of cells is at present an unresolved issue.

To study the possible mechanism by which peripheral nerves mediate immune responses in target tissues, electrical stimulation of the sciatic nerve was combined with subcutaneous microdialysis of the hind paw. Following unilateral stimulation of the sciatic nerve, an ipsilateral rise in substance P and a bilateral rise in VIP levels were observed in dialysate samples from experimental vscontrol animals. Electrical stimulation of the sciatic nerve induced a marked hyperemia and swelling of the ipsilateral paw. Quantitative immunocytochemical analysis of paraffin-embedded sections of the hind foot pads demonstrated T lymphocyte migration ipsilateral to the stimulated nerve. These findings suggest that peripheral nerves can directly modulate local immune and inflammatoryresponses.

Tissue factor (TF), a cell surface-associated cofactor and activator of coagulation factor VII, has been implicated in the local and systemic activation of coagulation associated with sepsis. This study describes the pattern of TF expression in experimental bovine pneumonic pasteurellosis and endotoxemia. Immunohistochemical techniques were used to localize TF antigen in tissue sections. Tissue factor expression was not observed in tissues from control animals. In response to Pasteurella haemolytica challenge, TF was expressed within alveolar walls, by mononuclear inflammatory cells within alveoli, and in walls of arteries, arterioles, bronchi, and bronchioles. Tissue factor was not detected in unaffected lung, liver, spleen, lymph node or kidney tissue. Administration of Escherichia coli endotoxin intravenously resulted in tissue factor expression in lung, spleen, and lymph node tissue. Results of this study indicate that TF is expressed locally at sites of inflammation and systemically in endotoxemia. Therefore, TF may be involved in coagulation events associated with pneumonic pasteurellosis.

A polymerase chain reaction (PCR) assay was used to confirm the presence of ileal symbiont (IS) intracellularis in 3 swine herds with a history of proliferative enteritis (PE). Two pooled fecal specimens, each comprising 5 individual stool samples, were collected from pen floors to screen for the presence of IS intracellularis and determine the age range of pigs shedding the organism. IS intracellularis was detected in the feces of clinically normal 10–25week-old grower/finisher pigs, indicating that this age range of pigs was the main source of infection for younger nursery pigs. Shedding continued without clinical disease when 10–100 g/ton of tylosin or 10 g/ton of chlortetracycline was added to the feed. PCR testing of pooled fecal samples can be used to identify groups of pigs affected with PE. The results of this study indicate that this PCR assay has the potential to accurately assess the IS intracellularis infection status of swine herds and the association of IS intracellular-is with PE and growth performance.

One-, 4-, and lo-week-old pigs were exposed to porcine reproductive and respiratory syndrome virus (PRRSV) to determine the effect of age on clinical signs, hematologic alterations, the onset and duration of viremia, routes of virus shedding, antibody production, and microscopic lesions produced by PRRSV isolate ATCC VR-2332. The response to PRRSV infection was similar among age groups. Fever, usually prolonged, and a marked dyspnea with cutaneous erythema when restrained for sample collection were the most consistent clinical signs. Prolonged periocular edema was unique to the 1-week-old pigs. The white blood cell count was decreased on day 4 postexposure (PE) due to decreases in neutrophils and lymphocytes. The virus was isolated from buffy coats at day 1 PE and was isolated from serum, buffy coat, or plasma at each sample collection period through the end of the trial (day 28 PE). Virus was most consistently isolated from lung, lymph node, spleen, and tonsil on day 7 PE and exclusively from lymph node, spleen, and tonsil on day 28 PE. Virus was infrequently isolated from urine and fecal and nasal swabs. Consistent microscopic changes in all age groups included interstitial pneumonia and lymph node hypertrophy and hyperplasia on days 7 and 28 PE, lymph node necrosis on day 7 PE, and subacute mononuclear myocarditis on day 28 PE. Findings presented here indicate that interstitial pneumonia, lymphoid necrosis, and mononuclear myocarditis are characteristic lesions of PRRSV isolate ATCC VR-2332 infection in 1-, 4-, and lo-week-old pigs.

The production of superoxide anion by NADPH oxidase is a principal nonspecific bactericidal activity of macrophages and neutrophils in host defense. However, exuberant production of superoxide anion also damages host tissues. Cloning and DNA sequencing of the 91 kDa subunit (gp91-phox) open reading frame indicated a high degree of sequence conservation, greater than 90% in nucleotide and amino acid sequences, between the porcine and human cDNAs. We show in pigs that interleukin-4 (IL-4), a T lymphocyte cytokine which plays a major role in mediating antibody responses to pathogens, suppresses superoxide anion production in macrophages by specifically reducing the level of mRNA encoding gp91-phox. Messenger RNA levels are suppressed approx. 70% within 4 h and persist for 24 h without any change in the rate of mRNA turnover. Nuclear run-on analysis showed that IL-4 did not alter the rate of gp91-phox gene transcription under conditions in which IL-1β transcription was inhibited. These results indicate that IL-4 suppresses the inflammatory response of macrophages by mechanisms that include post-transcriptional regulation of the 91 kDa catalytic subunit of NADPH oxidase, and transcriptional regulation of inflammatory cytokine expression.

Inflammatory cytokines, including interleukin (IL)‐1α, IL‐1β, IL‐8, and tumor necrosis factor α(TNF‐α) are produced by macrophages in response to a variety of pathogenic stimuli. We show here that the expression of inflammatory cytokines is suppressed by IL‐4 at the transcriptional level. Interleukin‐4, when added together with bacterial lipopolysaccharide (LPS), suppressed LPS‐induced increases in mRNA levels of IL‐1α, IL‐1β, IL‐8, and TNF‐αin alveolar macrophages. The level of suppression was dependent on dose and time of exposure and reached a maximum of 75–80% of uninduced values for IL‐1α, IL‐8, and TNF. Interleukin‐1βexpression was completely inhibited by IL‐4. The amount of secreted protein, as determined by TNF‐αbioassay, was also suppressed by IL‐4. Half‐maximal suppression occurred at IL‐4 concentrations between 0.02 and 0.1 ng/ml for all inflammatory cytokines. Nuclear run‐on assays showed that IL‐4 suppressed transcriptional activity of all inflammatory cytokines. Messenger RNA stability was not changed by IL‐4. The data suggest that IL‐4 plays an important transcriptional role in the regulation of alveolar macrophage inflammatory activities in respiratory disease and raise the possibility that IL‐4 may function in vivo as a coordinator of inflammatory and immune responses J. Leukoc. Biol.56: 507–513; 1994.

We report comparative linkage mapping of eleven genes in the swine genome by RFLP analysis. These genes include: Acid phosphatase type 5 (ACP5), Cholecystokinin Type B Receptor (CCKBR), Antibiotic Peptide (FALL39), Insulin-like Growth Factor 1 Receptor (IGF1R), Integrin Alpha M (ITGAM), Integrin Beta 2 (ITGβ2), Opioid Receptor Mu-1 (OPRM1), Prohormone Converter (PCI/3), Retinol Binding Protein 3 (RBP3), Ribosomal DNA (RNR1), and Zona Pellucida Glycoprotein 1 (ZP1). The CCKBRand ITGβ2 loci define the ends of the linkage groups on Chromosomes (Chro) (SSC) 9p and 13qter, respectively.

Factors that influence the development of adolescent autonomy were examined in a longitudinal study of 30 learning handicapped and 30 nonhandicapped adolescents. Autonomy was represented by three separate measures: distance from parental supervision, responsibility, and deviance. Findings suggest that handicapped adolescents lag behind their nonhandicapped peers in achieving separation from parental supervision, in part because of more restrictive parental rules. But the gap appears to be shrinking as the handicapped adolescents are making strides to catch up during the high school years. In both groups, the families where autonomy is achieved with the least amount of discord are those where parents relax control gradually during the period of adolescence.

Serological indicators of general condition would be helpful for monitoring or assessing ungulate wildlife. Toward that end, we report the 1st reference values for 2 cytokines, interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-a), in neonatal elk (Cervus elaphus). We obtained blood samples from 140 calves = 6 days old in Yellowstone National Park during summer 2003–2005. IL-6 values ranged from 0 to 1.21 pg/ml with a median of 0.03 pg/ml. TNF-a values ranged from 0 to 225.43 pg/ml with a median of 1.85 pg/ml. IL-6 and TNF-a concentrations were not significant predictors of elk calf survival through 21 days. Development of ungulate-based IL-6 and TNF-a assays that provide greater sensitivity than cross-reacting human-based assays could be helpful in monitoring ungulate condition and health status comparisons among herds. Such information could provide indirect assessments of range quality or environmental influences among herds.

Male spider mites of two sibling species, Tetranychus urticae and Tetranychus cinnabarinus, compete equally for and mate nonassortatively with females of either species. Fecundity in females mated interspecifically is normal but the female/male sex ratio of hybrid progeny is reduced. The ability of males to compete equally enhances the potential of hybrid sterility as a realistic genetic control method for spider mites in greenhouses.

Despite inherent difficulties in defining and measuring stress, a scientific framework has been provided in recent years for understanding how disruptive life experiences might be translated into altered susceptibility to infectious diseases. Studies of the effects of stress on pathogenesis of infectious disease are highly relevant to assessment of the biological importance of the immune impairments that have been associated with stress. With a few notable exceptions, investigations of viral infections in humans and in animal models support the hypothesis that stress promotes the pathogenesis of such infections. Similar conclusions can be drawn from studies of bacterial infections in humans and animals and from a small number of studies of parasitic infections in rodent models. While many of these studies have substantial limitations, the data nonetheless suggest that stress is a potential cofactor in the pathogenesis of infectious disease. Given recent unprecedented advances in the neurosciences, in immunology, and in the field of microbial pathogenesis, the relationship between stress and infection should be a fruitful topic for interdisciplinary research.