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Adjuvants enhance both the magnitude and duration of immune responses, therefore representing a central component of vaccines. The nature of the adjuvant can determine the particular type of immune response, which may be skewed toward cytotoxic T cell (CTL) responses, antibody responses, or particular classes of T helper (Th) responses and antibody isotypes. Traditionally, adjuvants have been added to intrinsically poor immunogenic vaccines, such as those using whole killed organisms or subunit vaccines. Here, we have compared cellular immune responses induced by the immunogenic modified life-attenuated vaccine Ingelvac PRRS® MLV when administered alone or in combination with carbopol, a widely used adjuvant in veterinary medicine. Using functional readouts (IFN-γ ELISpot and cell proliferation) and analyzing phenotypical hallmarks of CD4T cell differentiation, we show that carbopol improves cellular immunity by inducing early IFN-γ-producing cells and by preferentially driving T cell differentiation to effector phenotypes. Our data suggest that adjuvants may enhance and modulate life-attenuated--not only subunit/inactivated--vaccines., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

The family Anelloviridae includes human and animal torque teno viruses (TTVs) with extensive genetic diversity. The antigenic diversity among anelloviruses has never been assessed. Using torque teno sus virus (TTSuV) as a model, we describe here the first investigation of the antigenic relationships among different anelloviruses. Using a TTSuV genotype 1a (TTSuV1a) or TTSuV1b enzyme-linked immunosorbent assay (ELISA) based on the respective putative ORF1 capsid antigen and TTSuV1-specific real-time PCR, the combined serological and virological profile of TTSuV1 infection in pigs was determined and compared with that of TTSuV2. TTSuV1 is likely not associated with porcine circovirus-associated disease (PCVAD), because both the viral loads and antibody levels were not different between affected and unaffected pigs and because there was no synergistic effect of concurrent PCV2/TTSuV1 infections. We did observe a higher correlation of IgG antibody levels between anti-TTSuV1a and -TTSuV1b than between anti-TTSuV1a or -1b and anti-TTSuV2 antibodies in these sera, implying potential antigenic cross-reactivity. To confirm this, rabbit antisera against the putative capsid proteins of TTSuV1a, TTSuV1b, or TTSuV2 were generated, and the antigenic relationships among these TTSuVs were analyzed by an ELISA and by an immunofluorescence assay (IFA) using PK-15 cells transfected with one of the three TTSuV ORF1 constructs. The results demonstrate antigenic cross-reactivity between the two genotypes TTSuV1a and TTSuV1b but not between the two species TTSuV1a or -1b and TTSuV2. Furthermore, an anti-genogroup 1 human TTV antiserum did not react with any of the three TTSuV antigens. These results have important implications for an understanding of the diversity of anelloviruses as well as for the classification and vaccine development of TTSuVs.

Anelloviruses are a group of single-stranded circular DNA viruses infecting humans and other animal species. Animal models combined with reverse genetic systems of anellovirus have not been developed. We report here the construction and initial characterization of full-length DNA clones of a porcine anellovirus, torque teno sus virus 2 (TTSuV2), in vitro and in vivo. We first demonstrated that five cell lines, including PK-15 cells, are free of TTSuV1 or TTSuV2 contamination, as determined by a real-time PCR and an immunofluorescence assay (IFA) using anti-TTSuV antibodies. Recombinant plasmids harboring monomeric or tandem-dimerized genomic DNA of TTSuV2 from the United States and Germany were constructed. Circular TTSuV2 genomic DNA with or without introduced genetic markers and tandem-dimerized TTSuV2 plasmids were transfected into PK-15 cells, respectively. Splicing of viral mRNAs was identified in transfected cells. Expression of TTSuV2-specific open reading frame 1 (ORF1) in cell nuclei, especially in nucleoli, was detected by IFA. However, evidence of productive TTSuV2 infection was not observed in 12 different cell lines transfected with the TTSuV2 DNA clones. Transfection with circular DNA from a TTSuV2 deletion mutant did not produce ORF1 protein, suggesting that the observed ORF1 expression is driven by TTSuV2 DNA replication in cells. Pigs inoculated with either the tandem-dimerized clones or circular genomic DNA of U.S. TTSuV2 developed viremia, and the introduced genetic markers were retained in viral DNA recovered from the sera of infected pigs. The availability of an infectious DNA clone of TTSuV2 will facilitate future study of porcine anellovirus pathogenesis and biology.

Porcine Torque teno virus (TTV) has a single-stranded circular DNA genome and is currently classified into a new genus Iotatorquevirus with two species in a newly established family Anelloviridae. Viral DNA of both porcine TTV species (TTSuV1 and TTSuV2) has a high prevalence in both healthy and diseased pigs worldwide and multiple infections of TTSuV with distinct genotypes or subtypes of the same species has been documented in the United States and in Europe. However, the prevalence of specific TTSuV antibodies in pigs remains unknown. In this study, the putative ORF1 capsid protein from TTSuV2 isolate PTTV2c-VA was expressed in Escherichia coli. The purified recombinant ORF1 protein was used as the antigen for the development of Western blot and indirect ELISA to detect TTSuV2-specific IgG antibodies in pig sera. The results revealed a relatively high rate of seropositivity to TTSuV2 in conventional pigs from different sources but not in gnotobiotic pigs. Overall, pigs with undetectable TTSuV2 viral load were more likely to have a lower anti-TTSuV2 antibody level. An analysis of 10 conventional pigs during a 2-month period showed that decreased viral loads or presumed virus clearance were associated with elevated anti-ORF1 IgG antibody levels. Interestingly, porcine circovirus associated disease (PCVAD)-affected pigs had a significantly lower level of TTSuV2 antibody than PCVAD-unaffected pigs (p<0.01). This is the first study to establish essential serodiagnostic tools for investigation of TTSuV seroprevalence and infection dynamics, which will help elucidate the potential pathogenicity of TTSuV infection in pigs., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Porcine Torque teno virus (TTV), a single-stranded circular DNA virus, has been incriminated in swine diseases recently. Multiple infection with porcine TTV species 1 (PTTV1) and species 2 (PTTV2), each consisting of two types (PTTV1a and 1b) or subtypes (PTTV2b and 2c), in a single pig had been reported by our group previously. The present study described three novel assays for quantitation and differential detection of porcine TTV. First, we developed two SYBR green-based real-time PCR assays to quantify viral loads of two porcine TTV species, respectively. The PTTV1- and PTTV2-specific real-time PCR primer sequences were selected to target conserved regions identified by multiple alignments of ten available porcine TTV full-length genomes. Furthermore, by coupling the two singleplex PCR assays, a duplex real-time PCR assay followed by melting curve analysis was established for simultaneous detection and differentiation of PTTV1 and PTTV2. In addition, a type-specific duplex nested PCR was also developed to simultaneously detect and distinguish between the two types, PTTV1a and 1b, in PTTV1 species. These assays provide rapid and practical tools for molecular diagnosis of species- or type-specific porcine TTV., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The vaccine efficacy of six PRRSV Type 2 infectious clones, including five chimeras and a strain-specific deletion mutant, were examined using a respiratory challenge model in growing swine. The chimeras were constructed from different combinations of a licensed modified live vaccine (Ingelvac PRRS MLV) and a virulent field isolate (wt MN184) which differ by 14.3% on a nucleotide basis, while the deletion mutant tested had a broad deletion in the nsp2 region of strain MN184. The appearance of antibodies and virus characterization revealed regions of the genome that could influence PRRSV replication in vivo. Swine growth, clinical signs and lung lesions were also monitored. Average daily weight gain was negatively and directly impacted by some vaccines, and after challenge, vaccination with different constructs led to variable weight gain. We determined that 3 of the tested chimeras, including two previously published chimeras [1] and one in which strain MN184 ORF5-6 was placed on the background of Ingelvac PRRS MLV were able to prevent lung consolidation to a similar extent as traditionally prepared cell-passaged attenuated vaccines. The study suggested that only specific chimeras can attenuate clinical signs in swine and that attenuation cannot be directly linked to primary virus replication. Additionally, the strain MN184 deletion mutant was not found to have been sufficiently attenuated nor efficacious against heterologous challenge with strain JA-142., (Published by Elsevier Ltd.)

The stability of Porcine reproductive and respiratory syndrome virus (PRRSV) was evaluated for temperatures appropriate to laboratory and field settings. Four North American (type 2) isolates (ATCC VR-2332, JA-142, MN-184, and Ingelvac(R) PRRS ATP vaccine virus) in cell culture medium (pH 7.5) were held at 1 of 4 temperatures (4, 10, 20, and 30 degrees C) and sampled over time. Samples were tested for infectious virus and total PRRSV RNA using median tissue culture infectious dose and quantitative reverse transcription polymerase chain reaction, respectively. The rate of loss of infectious virus was expressed in terms of the time required for virus concentration to decline by one half (i.e., half-life [T(1/2)]). Statistical analysis found that temperature, but not virus isolate, had a significant effect on T(1/2), and a single nonlinear regression model was derived to predict T(1/2) for temperatures between 0 and 50 degrees C: T(1/2) = 243.54 e((-0.109*TEMP)). In contrast to changes over time in the concentration of infectious virus, no change in the concentration of quantitative reverse transcription polymerase chain reaction-detectable PRRSV was detected at any temperature and contact time. This information will be of interest to persons working in laboratory or field situations in which the control of PRRSV is important.

Background: Porcine reproductive and respiratory syndrome virus (PRRSV) causes a prolonged, economically devastating infection in pigs, and immune resistance to infection appears variable. Since the porcine adaptive immune system is not fully competent at birth, we hypothesized that age influences the dynamics of PRRSV infection. Thus, young piglets, growing 16-20-week-old finisher pigs, and mature third parity sows were infected with virulent or attenuated PRRSV, and the dynamics of viral infection, disease, and immune response were monitored over time., Results: Virulent PRRSV infection and disease were markedly more severe and prolonged in young piglets than in finishers or sows. Attenuated PRRSV in piglets also produced a prolonged viremia that was delayed and reduced in magnitude, and in finishers and sows, about half the animals showed no viremia. Despite marked differences in infection, antibody responses were observed in all animals irrespective of age, with older pigs tending to seroconvert sooner and achieve higher antibody levels than 3-week-old animals. Interferon gamma (IFN gamma) secreting peripheral blood mononuclear cells were more abundant in sows but not specifically increased by PRRSV infection in any age group, and interleukin-10 (IL-10) levels in blood were not correlated with PRRSV infection status., Conclusion: These findings show that animal age, perhaps due to increased innate immune resistance, strongly influences the outcome of acute PRRSV infection, whereas an antibody response is triggered at a low threshold of infection that is independent of age. Prolonged infection was not due to IL-10-mediated immunosuppression, and PRRSV did not elicit a specific IFN gamma response, especially in non-adult animals. Equivalent antibody responses were elicited in response to virulent and attenuated viruses, indicating that the antigenic mass necessary for an immune response is produced at a low level of infection, and is not predicted by viremic status. Thus, viral replication was occurring in lung or lymphoid tissues even though viremia was not always observed.

Two genetically distinct infectious recombinant virus clones (pMLV, constructed from Ingelvac PRRS MLV and pMN184, constructed from virulent strain MN184) were developed to study attenuation of contemporary porcine reproductive and respiratory syndrome virus (PRRSV) strain MN184. Two reciprocal chimeric clones (pMLVORF1/MN184 and pMN184ORF1/MLV) were then constructed, such that the 5'UTR/ORF1 of one genotype was linked to ORF2-7/3'UTR from the other genotype. In vitro studies demonstrated that the rescued chimeric viruses possessed intermediate growth properties compared to recombinant rMLV and rMN184. Swine inoculation with rMN184 and rMLV verified that these viruses fully mimicked the respective parent virus. In addition, earlier and higher antibody responses were detected in animals infected with rMN184 in contrast to those infected with rMLV. Chimeric virus treatment groups showed similar antibody responses as seen with these parent viruses, but much less severe pathogenesis when compared to the rMN184 group. These data suggested that genetic aspects of Ingelvac PRRS MLV 5'UTR/ORF1 replicase region and/or the structural proteins/3'UTR can serve to attenuate virulent strain MN184. The data also indicated that designed PRRSV vaccines could be developed, keeping the known 5'UTR/replicase region of an early vaccine strain such as Ingelvac PRRS MLV intact, but replacing the structural protein/3'UTR domain with that of an emerging virulent virus.

Proliferative enteropathy (PE; ileitis) is a common intestinal disease affecting susceptible pigs raised under various management systems around the world. Major developments in the understanding of PE and its causative agent, Lawsonia intracellularis, have occurred that have led to advances in the detection of this disease and methods to control and prevent it. Diagnostic tools that have improved overall detection and early onset of PE in pigs include various serological and molecular-based assays. Histological tests such as immunohistochemistry continue to be the gold standard in confirming Lawsonia-specific lesions in pigs post mortem. Despite extreme difficulties in isolating L. intracellularis, innovations in the cultivation and the development of pure culture challenge models, have opened doors to better characterization of the pathogenesis of PE through in vivo and in vitro L. intracellularis-host interactions. Advancements in molecular research such as the genetic sequencing of the entire Lawsonia genome have provided ways to identify various immunogens, metabolic pathways and methods for understanding the epidemiology of this organism. The determinations of immunological responsiveness in pigs to virulent and attenuated isolates of L. intracellularis and identification of various immunogens have led to progress in vaccine development.

At the most elemental level, the design of effective strategies to control and/or eliminate porcine reproductive and respiratory syndrome (PRRS) virus depend on an accurate and comprehensive understanding of virus transmission. As a general rule, transmission is highly dependent on the route of exposure and the dose of virus. The objective of this study was to derive PRRS virus isolate VR-2332 dose-response curves for oral and intranasal routes of exposure, i.e., determine the probability that a specific virus dose would result in infection. Individually housed pigs approximately 21 days of age were exposed to specific doses of PRRS virus isolate VR-2332 by either oral or intranasal routes. Positive controls were intramuscularly inoculated with 10(2.2) 50% tissue culture infective dose (TCID50) of PRRS virus and negative controls were orally administered 100ml of diluent with no virus. Pigs were monitored for evidence of infection for 21 days following exposure, i.e., serum samples were collected on days 0, 7, 14, 21, and tested for virus and PRRS virus-specific antibodies. Dose-response curves and 95% confidence intervals for oral and intranasal routes of exposure were derived using logistic models (logit and probit). The infectious dose50 (ID50) for oral exposure was estimated to be 10(5.3) TCID50 (95% CI, 10(4.6) and 10(5.9)); the ID50 for intranasal exposure was estimated to be 10(4.0) TCID50 (95% CI, 10(3.0) and 10(5.0)). Given these estimates, it is worth noting that intramuscular exposure of animals to 10(2.2) TCID50 (positive controls) resulted in infection in all animals. Thus pigs were the most susceptible to infection via parenteral exposure.

An enzyme-linked immunosorbent assay (ELISA) for Lawsonia intracellularis was developed and compared with a whole-cell antigen-based immunofluorescence antibody test (IFAT). The antigen-containing lipopolysaccharide (LPS) was derived from Percoll gradient purified cultures of L. intracellularis by using a modification of the Westphal hot phenol procedure. The antigen was bound directly to polystyrene 96-well microtiter plates, and the assay was performed in an indirect ELISA format. Specificity and sensitivity values based on 80 known positive and 80 known negative serum samples from controlled experimental trials were 93.7% and 88.7%, respectively. Serological results from a controlled L. intracellularis challenge exposure study confirmed the high specificity and sensitivity of this assay (100% and 99.5%, respectively). Comparisons between the LPS ELISA and the IFAT in detecting anti-Lawsonia antibodies in this controlled study revealed significantly more LPS ELISA-positive pigs than IFAT-positive pigs on days 21, 28, 35, and 42 (P = 0.003, 0.030, 0.002, and 0.006, respectively). This indirect ELISA (LPS ELISA) test is an improved method of detecting antibodies in pigs soon after exposure to L. intracellularis, regardless of isolate type (vaccine or wild type) in experimental studies. The LPS ELISA may be used as a tool to support future research trials on vaccine efficacy and to further understand the immune response induced by L. intracellularis.

Transmission of Lawsonia intracellularis from experimentally inoculated pigs to naive swine was demonstrated in this study. The study was conducted using conventional pigs divided into three groups as follows: principles inoculated with L. intracellularis, sentinels, and controls. The pigs were inoculated and paired on 13 and 9 days post-inoculation with a sentinel pig for 7 days. Fecal samples and serum samples were collected throughout the study for polymerase chain reaction (PCR) and antibody testing by indirect fluorescent antibody techniques. After co-mingling, the inoculated group was necropsied; sentinel and control pigs were necropsied 7-14 days later. The intestinal tracts were evaluated grossly and microscopically for lesions. PCR was performed on intestinal mucosal scrapings and feces. Warthin-Starry and fluorescent antibody staining procedures were conducted to confirm colonization with L. intracellularis. Gross and microscopic lesions typical of porcine proliferative enteropathy (PPE) were observed in both the inoculated and sentinel groups. Transmission was demonstrated from inoculated principle pigs to sentinel pigs. PCR results detected cyclical shedding of L. intracellularis in the feces. Seroconversion occurred in pigs that were exposed to L. intracellularis. From this study, it was demonstrated that transmission of L. intracellularis can occur easily in an environment with experimentally infected pigs and that PCR can be a useful tool to monitor fecal shedding of the organism.

Objective: To evaluate the efficacy of an orally administered avirulent live vaccine to protect pigs against challenge exposure with virulent Lawsonia intracellularis., Animals: 108 weaned 3-week-old pigs (35 in experiment 1 and 73 in experiment 2)., Procedure: 2 experiments were conducted. On day 0, vaccinates were orally administered vaccine via drench or in drinking water, whereas challenge-control pigs were administered cultured medium. On day 21, pigs were challenge exposed with a virulent heterologous isolate of L. intracellularis. Clinical observations, weights, seroconversion, and fecal excretion of L. intracellularis were measured until day 42. At study termination, pigs were euthanatized and examined for L. intracellularis-specific lesion development of the ileum and colon., Results: Pigs receiving a single dose of vaccine were protected when challenge exposed with virulent L. intracellularis (at least 10(77) TCID50/dose). In experiment 1, vaccinates had significantly less fecal excretion (47% and 40% for days 35 and 42, respectively), compared with challenge-control pigs. In experiment 2, vaccinates had significantly less fecal excretion (50% and 58% for days 35 and 42, respectively), compared with challenge-control pigs. Significant reductions in lesion development were evident in the ileum of vaccinated pigs (70% and 56% at day 42 for experiments 1 and 2, respectively), compared with challenge-control pigs., Conclusions and Clinical Relevance: Oral administration by drench or via drinking water of an avirulent live vaccine against L. intracellularis resulted in substantial protection against proliferative enteropathy among vaccinates and offers a better way to reduce stress of pigs during vaccine administration.

Replacement of the carboxylic acid group of PGF(2alpha) with the non-acidic substituents hydroxyl (-OH) or methoxy (-OCH(3)) resulted in an unexpected activity profile. Although PGF(2alpha) 1-OH and PGF(2alpha) 1-OCH(3) exhibited potent contractile effects similar to 17-phenyl PGF(2alpha) in the cat lung parenchymal preparation, they were approximately 1000 times less potent than 17-phenyl PGF(2alpha) in stimulating recombinant feline and human FP receptors. In human dermal fibroblasts and Swiss 3T3 cells PGF(2alpha) 1-OH and PGF(2alpha) 1-OCH(3) produced no Ca(2+) signal until a 1 microM concentration was exceeded. Pretreatment of Swiss 3T3 cells with either 1 microM PGF(2alpha) 1-OH or PGF(2alpha) 1-OCH(3) did not attenuate Ca(2+) signal responses produced by PGF(2alpha) or fluprostenol. In the rat uterus, PGF(2alpha) 1-OH was about two orders of magnitude less potent than 17-phenyl PGF(2alpha) whereas PGF(2alpha) 1-OCH(3) produced only a minimal effect. Radioligand binding studies on cat lung parenchymal plasma membrane preparations suggested that the cat lung parenchyma does not contain a homogeneous population of receptors that equally respond to PGF(2alpha)1-OH, PGF(2alpha)1-OCH(3), and classical FP receptor agonists. Studies on smooth muscle preparations and cells containing DP, EP(1), EP(2), EP(3), EP(4), IP, and TP receptors indicated that the activity of PGF(2alpha) 1-OH and PGF(2alpha) 1-OCH(3) could not be ascribed to interaction with these receptors. The potent effects of PGF(2alpha) 1-OH and PGF(2alpha) 1-OCH(3) on the cat lung parenchyma are difficult to describe in terms of interaction with the FP or any other known prostanoid receptor.

Objective: To determine stability of the restriction fragment length polymorphism (RFLP) pattern of a porcine reproductive and respiratory syndrome vaccine virus and patterns of other viral strains as they replicate in pigs., Sample Population: Field samples of porcine reproductive and respiratory syndrome virus (PRRSV) and samples from 2 weaned pigs, 2 nursery-age pigs, and 5 gilts experimentally infected with PRRSV., Procedure: PRRSV was isolated from field samples, experimentally infected pigs, or pigs that were in contact with experimentally infected pigs. For each virus, RNA was isolated from infected cells, and RFLP patterns were determined., Results: 61% of field samples had 2-5-2 RFLP patterns characteristic of the vaccine virus, 32% had field virus RFLP patterns, and 7% had intermediate RFLP patterns that indicated a virus with a close relationship to the vaccine virus. Viruses isolated from experimentally infected pigs had no change in RFLP patterns after up to 13 weeks of in vivo replication and transmission to contact pigs., Conclusions and Clinical Relevance: RFLP patterns distinguish the vaccine and field strains of PRRSV; however, as the vaccine virus spreads among a swine population, the RFLP pattern can change to a related intermediate pattern. A glycine at residue 151 of open reading frame 5 is another marker for the vaccine virus; this glycine is rapidly lost and eventually replaced with arginine as the vaccine virus replicates in pigs.

Objective: To determine the predominant strain of progeny virus in samples obtained from cell cultures and pigs exposed simultaneously to attenuated and virulent strains of porcine reproductive and respiratory syndrome virus (PRRSV)., Sample Population: Cell cultures and twenty 4-week-old pigs., Procedure: Cell cultures and pigs were simultaneously exposed to various relative concentrations of an attenuated, cell-culture-adapted vaccine strain and a virulent field strain of PRRSV. Progeny virus obtained at selected intervals thereafter was tested to determine strain identity by use of restriction fragment length polymorphism (RFLP) analysis., Results: Progeny virus from infected cell cultures comprised the attenuated strain, alone or in combination with the virulent strain, except when cultures had been exposed to a large excess (> 100,000-fold) of the virulent strain. Progeny virus from infected pigs comprised only the virulent strain regardless of the relative concentrations of the 2 strains to which the pigs had been exposed., Conclusions: During concurrent replication in cell cultures, the attenuated strain quickly predominated. Conversely, during concurrent replication in pigs, the virulent strain quickly predominated., Clinical Relevance: It is unlikely that only an attenuated strain of PRRSV would be identified by RFLP testing of samples obtained from pigs concurrently infected with a virulent strain of PRRSV. Nevertheless, the ability of a cell-culture-adapted attenuated strain of PRRSV to predominate during cell culture passage (the first step in the current RFLP testing procedure) indicated that, if possible, samples should be obtained from pigs that do not have a history of direct or indirect exposure to attenuated-virus vaccine.

The polymerase chain reaction (PCR) was evaluated for its usefulness as a diagnostic tool to detect Lawsonia (ileal symbiont) intracellularis. Porcine ilea were collected from swine cases submitted to the Iowa State University Veterinary Diagnostic Laboratory between December 1, 1994, and June 30, 1995. Sampling was random, with no regard to health status. There were 621 ileum scrapings evaluated using the PCR technique. Thirty-five of the samples were positive, either by PCR or conventional diagnostic methods such as histology and Warthin-Starry silver stain. These 35 samples were further evaluated by Warthin-Starry silver stain and indirect immunofluorescent antibody test (IFAT) to confirm the presence of L. intracellularis in the tissue sections. Of the 26 samples positive by PCR, 22 were positive by IFAT. Sixteen of the 22 were also positive when stained with Warthin-Starry and evaluated microscopically for typical bacteria. Nine of the original samples were negative by all 3 techniques. PCR appears more sensitive and specific for L. intracellularis detection than Warthin-Starry stain and IFAT. This study provides evidence that PCR may be useful as a reference standard for the detection of L. intracellularis. PCR may be an appropriate monitoring tool for swine herds because it is a rapid procedure that could be applied to batch testing. Although the test is currently too laborious and expensive for routine diagnostic use, there may be situations in which it is justified because of the advantages of greater sensitivity and specificity.

Objective: To evaluate polymerase chain reaction (PCR) for detection of Lawsonia intracellularis DNA in feces and an indirect fluorescent antibody test (IFAT) for detecting serum IgG antibodies in pigs exposed to L intracellularis., Animals: 15 seven-week-old pigs and 42 three-week-old pigs., Procedure: During 3 experiments, 23 pigs were inoculated with a pure culture of L intracellularis, 31 pigs served as noninoculated controls, and 3 pigs were used as sentinels. Fecal shedding of L intracellularis was monitored by use of PCR analysis at 7-day intervals. At euthanasia, the ileum was obtained for PCR and histologic analyses. Serum was obtained at 7-day intervals for use in the IFAT., Results: Polymerase chain reaction analysis detected L intracellularis DNA in the feces of 39% of the inoculated pigs; by postinoculation days 21 to 28, 90% of inoculated pigs developed IgG antibodies detected by IFAT. Neither L intracellularis DNA nor IgG antibodies were detected in any of the noninoculated control pigs at euthanasia. Sera from pigs inoculated with enteric pathogens other than L intracellularis did not contain detectable antibodies that reacted with L intracellularis by use of the IFAT., Conclusion: The IFAT for L intracellularis IgG antibody detection appeared to be a more sensitive antemortem test for detecting pigs experimentally infected with L intracellularis than was a PCR method for direct detection of the organism in the feces., Clinical Relevance: Not all animals that are infected with L intracellularis shed the organism in feces at detectable amounts.

Objective: To evaluate the safety and efficacy of avirulent live Salmonella choleraesuis strain 54 (SC54) as a vaccine to protect calves against salmonellosis caused by S dublin., Animals: 40 head of clinically normal 3 to 5-week-old male Holstein calves that were culture negative for Salmonella sp., Procedure: Calves were randomly assigned to 4 test groups of 10 calves each. Group 1 received 8.5 x 10(7) colony-forming units (CFU) of SC54 SC. Groups 2 and 3 received 1.13 x 10(9) CFU of SC54, SC and intranasally, respectively. Group 4 received saline solution as a vaccine control. All calves were challenge exposed orally with 1.74 x 10(9) CFU of virulent S dublin 14 days after vaccination. Clinical signs and Salmonella shedding were monitored for 28 days after vaccination. Calves were necropsied, and organs were cultured for Salmonella sp 14 days after challenge exposure., Results: Calves of groups 2 and 3 had slightly high rectal temperature after vaccination. Salmonella dublin challenge exposure resulted in mild clinical signs of salmonellosis. All vaccinated groups had significantly (P < 0.05) lower rectal temperature, fecal shedding of S dublin, and recovery of S dublin from organs after necropsy. SC54 was not recovered from fecal or blood samples collected after vaccination or from injection site samples or organs collected at necropsy., Conclusions: SC54 given intranasally or SC to calves was safe and significantly (P < 0.05) reduced clinical signs and bacterial shedding after oral challenge exposure with S dublin., Clinical Relevance: SC54 has potential as an effective vaccine to aid in prevention of salmonellosis caused by S dublin in calves.

1. The pharmacological activity of a novel series of 9,11-cyclic carbonate derivatives of prostaglandin F2 alpha (PGF2 alpha) was investigated in various isolated smooth muscle preparations possessing different prostanoid receptor subtypes as well as in human platelets. Since subdivision of thromboxane (TP-) receptors into vascular/smooth muscle and platelet subtypes is a controversial subject, our studies included a human smooth muscle preparation (myometrium) in addition to the widely used rat aorta and human platelets as TP-receptor preparations. 2. Two members of that series, AGN191976 and AGN192093 were found to be highly potent and selective thromboxane-mimetics. AGN191976 and AGN192093 contracted isolated tissues of the rat thoracic aorta with EC50 values of 0.32 +/- 0.08 and 1.30 +/- 0.53 nM, respectively. Both agonists were at least 10 times more potent than the benchmark TP-agonist, U-46619, in this preparation, whilst being at least 500 times less potent at other prostanoid receptors (DP, EP1, EP3, FP, IP) in vitro. 3. In human myometrial strips from pregnant and non-pregnant donors, both AGN191976 and AGN192093 were potent contractile agonists. The rank order of potency in myometrium of AGN191976 > AGN192093 > U-46619 correlated well with that in the rat aorta. In human platelet-rich plasma (PRP), however, AGN191976 had potent proaggregatory activity (EC50 = 16.3 +/- 1.4 nM), which is a TP-receptor-mediated event, whereas AGN192093 was a much weaker agonist (EC50 = 37.9 +/- 2.0 microM). AGN192093 did not behave as an antagonist in the platelets, since it did not antagonize platelet aggregation induced by ADP, arachidonic acid, U-46619 or AGN191976. In human washed platelets, the activity profile of AGN191976 (EC50 = 4.15 +/- 0.52 nM) and AGN192093 (no aggregation up to 10 microM) was similar to that obtained in PRP. 4. The involvement of TP-receptors was verified with the potent TP-antagonist, SQ29548. SQ29548 (0.1 microM in myometrium; 1 microM in aorta; 1 microM and 10 microM in platelets) antagonized responses to U-46619, AGN191976 and AGN192093 as expected. 5. In conclusion, AGN191976 and AGN192093, both 9,11-cyclic carbonate derivatives of PGF2 alpha, were found to be highly potent and selective thromboxane-mimetics in rat vascular and human myometrial smooth muscle. However, only AGN 191976 was a potent agonist at TP-receptors in human platelets. The differential activity of AGN192093 on TP-receptor-mediated events in platelets and smooth muscle provides further evidence for a subdivision of TP-receptors. AGN192093 appears to be a useful tool for the pharmacological distinction of TP-receptor subtypes.

An avirulent live Salmonella choleraesuis culture (SC-54) was evaluated for use as an effective vaccine in preventing salmonellosis caused by S. choleraesuis in pigs. Eighty-two pigs, 3 to 4 weeks old, were randomly assigned to 1 of 2 treatment groups, which were designated as either vaccinates or controls. After vaccination, all pigs were examined for fecal shedding of S choleraesuis, rectal temperature, and 10 clinical variables. Significant difference was not detected between vaccinated and nonvaccinated pigs for 14 days (phase I) after intranasal administration of the vaccine. Efficacy and duration of immunity were examined by intranasally challenge exposing respective pigs from either treatment group with a virulent field isolate of S choleraesuis at 2, 8, or 20 weeks after vaccination (phases II-IV). Pigs were again evaluated for 14 days after challenge exposure, and 10 clinical variables and rectal temperature were monitored. Surviving pigs were euthanatized and evaluated for gross lesions, and samples of 7 organs were collected. These organs samples were homogenized, and level of S choleraesuis infection was determined. After virulent challenge exposure during phases II-IV, the clinical status of the SC-54 vaccinates was significantly (P < 0.05) superior to that of nonvaccinates for rectal temperature, feces consistency, behavior, appetite, body condition, and mean score for the 10 clinical variables. Quantitative bacteriologic culture of the tonsil, lung, liver, spleen, mesenteric lymph nodes, ileum, and colon samples indicated consistent reduction of organ colonization in vaccinates; bacteria numbers in the mesenteric lymph nodes, lungs, and ileum were significantly (P < 0.05) reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel series of prostaglandin F2 alpha (PGF2 alpha) prodrugs, with acyl ester groups at the 9, 11, and 15 positions, was prepared in order to design clinically acceptable prostaglandins for treating glaucoma. Studies involving isolated esterases and ocular tissue homogenates indicated that 9-acyl esters cannot provide a prodrug since PGF2 alpha would not be formed as a product. In contrast, 11-mono, 15-mono, and 11, 15-diesters were converted to PGF2 alpha in ocular tissues and could, therefore, be considered as prodrugs of PGF2 alpha. Carboxylesterase (CE) appeared critically important for the hydrolytic conversion of those PGF2 alpha prodrugs where the 11 or 15-OH group was esterified and such prodrugs were not substrates for acetylcholinesterase (ACHE) or butyrylcholinesterase (BuCHE). The enzymatic hydrolysis of PGF2 alpha-1-isopropyl ester was also investigated for comparative purposes. This PGF2 alpha prodrug was a good substrate for CE, but was also hydrolysed by BuCHE, albeit at a much slower rate. The most striking feature of the enzymatic hydrolysis of PGF2 alpha-1-isopropyl ester in ocular tissue homogenates was that it was much faster than for prodrugs esterified at the 11 and/or 15 positions. In terms of ocular hypotensive activity, all prodrugs which showed detectable conversion to nascent PGF2 alpha were potent ocular hypotensives. Although no separation of ocular hypotensive and ocular surface hyperaemic effects was apparent for PGF2 alpha-1-isopropyl ester, a temporal separation of these effects was apparent for the novel PGF2 alpha ester series. This difference may reflect an unfavourably rapid conversion of PGF2 alpha-1-isopropyl ester in ocular surface tissues compared with anterior segment tissues.

Seventy-five pigs from 4 facilities were examined for Salmonella choleraesuis by use of bacteriologic culture of feces, blood, WBC (buffy coat), mononuclear leukocytes, and neutrophils. The organism was isolated from 0 of 75 fecal samples, compared with isolation from 39 of 75 purified neutrophil preparations. Of the pigs that did not have Salmonella isolated from feces or blood, but had S choleraesuis isolated from neutrophils, 6 were further examined. These pigs from 2 groups again had culture performed at least 3 successive times to test for repeatability and to determine optimal number of neutrophils required for Salmonella isolation. These same pigs were euthanatized and necropsied. Nineteen tissue specimens from each pig were obtained for culture, but S choleraesuis was isolated only from neutrophil samples. Results indicate that neutrophils may contribute to the carrier state in pigs and should be cultured when attempting to identify S choleraesuis carrier swine.

Salmonella choleraesuis strain 38 (glycerol-positive fermentation) was repeatedly exposed to porcine neutrophils in an attempt to mimic in vivo conditions of the host immune system. After phagocytosis, viable intracellular S choleraesuis were isolated and the process was repeated at least 5 times. A fifth-passage strain-38 neutrophil-adapted clone, 38PMNa-5X, was isolated, and was compared with the parent wild-type strain 38 for changes. Strain 38PMNa-5X had increased resistance to killing by hydrogen peroxide and phagocyte killing by porcine neutrophils, as measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction. Strain 38PMNa-5X was less invasive than the parent strain on Vero cell monolayers, and had been cured of a 50-kb plasmid. The 50-kb plasmid was marked with bacteriophage mini-Mu (kanamycin resistant) and was reinserted into strain 38PMNa-5X. Strain 38PMNa-5X was avirulent in mice, but the isolates with reinserted plasmids had intermediate resistance to neutrophil and hydrogen peroxide killing and had restored invasiveness and mouse virulence. Differences in complement sensitivity and enzymatic activity were not observed between the strains.

The purpose of this study was to determine the safety and efficacy of a live Salmonella choleraesuis immunizing strain, obtained by repeated ingestion and recovery through porcine neutrophils. The strain was tested in mice and in pigs. The vaccine was safe and effective in controlled experimental trials, using clinical, pathologic, and microbiologic criteria. Vaccinated pigs were able to maintain normal weight gains during the 4-week observation period following challenge inoculation with a high dose of a virulent strain.

The mechanisms of invasion used by virulent and avirulent Salmonella choleraesuis were compared using a Vero cell invasion assay. Mouse virulent S. choleraesuis strain 38 and avirulent strain 9 were examined for their ability to invade and survive in Vero cells. The assay was performed by S. choleraesuis infection of the Vero cell monolayer alone and in the presence of various treatments applied to the Vero cell monolayers. Intracellular S. choleraesuis colony forming units were then counted to characterize the mechanism of bacterial uptake. Invasion was not affected by colchicine, but was significantly inhibited in the presence of cytochalasins B and D, chloroquine, and dansylcadaverine. Inhibition by the above substances suggested the importance of microfilaments and of receptor recycling in receptor mediated endocytosis. Both bacterial strains had decreased invasion in the presence of mannose and after enzymic treatment with trypsin. Mannose exposure caused a significant 48% decrease in the uptake of virulent S. choleraesuis 38 and a 28% decrease in avirulent S. choleraesuis 9. Inhibition of endosome acidification did not affect the virulent strain 38 as much as it affected avirulent strain 9. Results from these experiments suggested that Vero cell invasion by S. choleraesuis was due to host uptake by receptor mediated endocytosis, and was mediated in part by mannose-sensitive adhesins. Outer membrane proteins were extracted from the virulent and avirulent strain and compared using SDS-PAGE following surface protein labeling with 125I. Virulent S. choleraesuis 38 had a unique 35 kD protein. The outer membrane proteins of both strains were then examined by radio-immunoprecipitation and western blot using guinea pig polyclonal antisera and the 35 kD protein was again found to be unique to the virulent strain 38. Antisera against the 35 kD protein significantly inhibited invasion of Vero cells by S. choleraesuis strain 38.

Porcine polymorphonuclear leukocytes (PMNLs) may be activated by bacteria to begin phagocytosis followed by oxidative and non-oxidative mechanisms of killing. The purpose of this study was to identify differences between virulent and avirulent Salmonella choleraesuis (S. choleraesuis) strains, 38 and 9 respectively, in their interactions with porcine PMNLs using five different assays. (1) Staphylococcus aureus (S. aureus) ingestion was determined by exposure of porcine PMNLs to a mixture of S. choleraesuis and 125I labeled S. aureus. There was a 2.98% and 22.20% decrease in S. aureus ingestion by mouse-avirulent S. choleraesuis 9 and mouse-virulent S. choleraesuis 38 respectively. (2) Iodination of proteins was done by exposing zymosan stimulated porcine PMNLs to S. choleraesuis in the presence of 125I and measuring its incorporation into porcine PMNL proteins. This assay indicated a 73.7% and 74.7% decrease in iodination by S. choleraesuis 9 and S. choleraesuis 38, respectively. (3) Cytochrome c reduction was performed by using porcine PMNLs, zymosan, and S. choleraesuis to determine the bacterial effect on superoxide anion production. S. choleraesuis 9 and S. choleraesuis 38 inhibited superoxide anion production by 78.0% and 92.6%, respectively. (4) Lactoferrin release from porcine PMNLs was measured by an ELISA using the supernatant from the cytochrome c assay. Results indicate a 52.0% and 61.0% increase in lactoferrin release by S. choleraesuis 9 and 38 respectively. (5) The bactericidal assay was performed by counting cfus of S. choleraesuis after preliminary incubation with porcine PMNLs, followed by killing of extracellular S. choleraesuis and lysis of porcine PMNLs. Survival of S. choleraesuis 9 and E. coli (control) were 7.50% and 1.37%, respectively, in contrast to 52.62% survival of the virulent S. choleraesuis 38. These results indicate that both strains inhibited protein iodination and caused a slight increase in lactoferrin release, but the virulent S. choleraesuis 38 inhibited S. aureus ingestion, cytochrome c reduction, and survived porcine PMNL killing more effectively than the avirulent S. choleraesuis 9.