A nanobody-horseradish peroxidase fusion protein-based competitive ELISA for rapid detection of antibodies against porcine circovirus type 2.

BACKGROUND: The widespread popularity of porcine circovirus type 2(PCV2) has seriously affected the healthy development of the pig industry and caused huge economic losses worldwide. A rapid and reliable method is required for epidemiological investigation and evaluating the effect of immunization. However, the current methods for PCV2 antibody detection are time-consuming or very expensive and rarely meet the requirements for clinical application. we have constructed the platform for expressing the nanobody(Nb)­horseradish peroxidase(HRP) fusion protein as an ultrasensitive probe to detect antibodies against the Newcastle disease virus(NDV), previously. In the present work, an Nb-HRP fusion protein-based competitive ELISA(cELISA) for rapid and simple detection antibodies against PCV2 was developed using this platform to detect anti-PCV2 antibodies in clinical porcine serum. RESULTS: Using phage display technology, 19 anti-PCV2-Cap protein nanobodies were screened from a PCV2-Cap protein immunized Bactrian camel. With the platform, the PCV2-Nb15­HRP fusion protein was then produced and used as a sensitive reagent for developing a cELISA to detect anti­PCV2 antibodies. The cut­off value of the cELISA is 20.72 %. Three hundreds and sixty porcine serum samples were tested by both newly developed cELISA and commercial kits. The sensitivity and specificity were 99.68 % and 95.92 %, respectively. The coincidence rate of the two methods was 99.17 %. When detecting 620 clinical porcine serum samples, a good consistent (kappa value = 0.954) was found between the results of the cELISA and those of commercial kits. CONCLUSIONS: In brief, the newly developed cELISA based PCV2-Nb15­HRP fusion protein is a rapid, low-cost, reliable and useful nanobody-based tool for the serological evaluation of current PCV2 vaccine efficacy and the indirect diagnosis of PCV2 infection.

Assessment of three commercial ELISAs for the detection of antibodies against Porcine epidemic diarrhea virus at different stages of the immune response.

Three commercial ELISAs -two based on spike (E1 and E3) and one on nucleocapsid protein (E2)-were used to analyze the development and persistence of antibodies against Porcine epidemic diarrhea virus (PEDV). Seventy-five four-week-old PEDV-negative piglets were inoculated orally with a European G1b PEDV (INOC) and fourteen were kept as controls (CTRL). After the inoculation, E3 detected positive animals as soon as 7 days post inoculation (dpi), while the earliest detection with E1 and E2 was at 14 dpi. All samples were positive at 21 and 28 dpi using E1 and E3, respectively, while E2 failed to detect 23.3 % of the inoculated pigs at any time point. The percentages of positive samples were different through the study: E1 and E3 > E2 from 14 to 56 dpi; and E3 > E1 > E2 from 56 to 154 dpi (P < 0.05). Five months after the inoculation, E3 still detected 92.0 % (IC95 % = 85.1-98.8 %) of pigs as positive, while E1 and E2 detected only 27.0 % (IC95 % = 16.0-37.9 %) and 0%, respectively. The sensitivity for E2 never exceeded 0.62. Specificity was 1 for all ELISAs. These different outcomes could be related to the ELISA strategies (indirect versus competition), the antigens used, the cut-off, or to other intrinsic factors of each test. The observed differences could be of importance when assessing whether older animals, such as fatteners or gilts, had previously been in contact with PEDV.

Generation and immunogenicity assessment of ELPylated virus-like particles of porcine circovirus type 2.

BACKGROUND: Porcine circovirus type 2 (PCV2) is an economically important pathogen affecting swine industry worldwide. The production of current PCV2 vaccines is time-consuming and expensive. Elastin-like polypeptides (ELP) undergo temperature-dependent inverse phase transition and ELPylated proteins can be purified simply by inverse transition cycling (ITC). METHODS: The Cap protein of PCV2b, together with the virus neutralizing (VN) epitopes of PCV2a, PCV2d and PCV2e, was expressed in E. coli as an ELPylated protein, and purified by ITC in the presence of mild detergents. For the control purpose, the Cap protein was also expressed as a His-tagged protein and purified by nickel affinity chromatography. The formation of ELPylated VLP (ELP-VLP) and His-tagged VLP (VLP) was revealed by transmission electron microscopy. Mice were immunized two times with the two forms of VLP and the antigen-specific IgG antibody, VN antibody, cytokine responses and immunoprotection against PCV2 challenge were compared. RESULTS: ELPylated Cap protein was expressed as a soluble protein and purified to 94.3% purity by ITC in the presence of 1% Triton X-100 and 0.5 M urea. His-tagged Cap fusion protein was expressed as insoluble inclusion bodies and purified to 90% purity under denatured conditions. The two purified fusion proteins assembled into VLP with similar morphology. Compared to immunization with VLP, immunization with ELP-VLP induced significantly (p < 0.01) stronger VN antibody response and slightly (p < 0.05) stronger Cap-specific IgG antibody response, cytokine production and immunoprotection against PCV2 challenge. CONCLUSION: A novel ELPylation platform for easy preparation of PCV2 VLP was established and the prepared ELP-VLP was more immunogenic than VLP. The ELPylation technology could be used for other VLP preparation and the prepared ELP-VLP could be developed as a novel PCV2 subunit vaccine.

The role of viral particle integrity in the serological assessment of foot-and-mouth disease virus vaccine-induced immunity in swine.

The efficacy of foot-and-mouth disease virus (FMDV) inactivated vaccines is mainly dependent on the integrity of the whole (146S) viral particles. If the intact capsids disassemble to 12S subunits, antibodies against internal-not protective epitopes, may be induced. Serological correlates with protection may be hampered if antibodies against internal epitopes are measured. Here we compared the performance of different ELISAs with the virus-neutralization test (VNT) that measures antibodies against exposed epitopes. Sera from pigs immunized with one dose of an expired commercial FMDV vaccine were used. This vaccine contained about 50% of O1/Campos and over 90% of A24/Cruzeiro strains total antigen as whole 146S particles. Specific-total antibodies were measured with the standard liquid-phase blocking ELISA (LPBE). We also developed an indirect ELISA (IE) using sucrose gradient purified 146S particles as capture antigen to titrate total antibodies, IgM, IgG1 and IgG2. A good correlation was found between VNT titers and IgG-ELISAs for A24/Cruzeiro, with the lowest correlation coefficient estimated for IgG2 titers. For O1/Campos, however, the presence of antibodies against epitopes different from those of the whole capsid, elicited by the presence of 12S particles in the vaccine, hampered the correlation between LPBE and VNT, which was improved by using purified O1/Campos 146S-particles for the liquid-phase of the LPBE. Interestingly, 146S particles but not 12S were efficiently bound to the ELISA plates, confirming the efficiency of the IE to detect antibodies against exposed epitopes. Our results indicate that any serological test assessing total antibodies or IgG1 against epitopes exposed in intact 146S-particles correlate with the levels of serum neutralizing antibodies in vaccinated pigs, and might potentially replace the VNT, upon validation. We recommend that antigen used for serological assays aimed to measure protective antibodies against FMDV should be controlled to ensure the preservation of 146S viral particles.

Assessment of hemagglutination activity of porcine deltacoronavirus.

Porcine deltacoronavirus (PDCoV) is an emerging swine enteric coronavirus that causes diarrhea in piglets. However, the biological characteristics of PDCoV are unclear. In this study, the hemagglutination (HA) abilities of two PDCoV strains (CH-01 and HNZK-04) were investigated. Our results showed that PDCoV has the ability to agglutinate rabbit erythrocytes after virion pretreatment with trypsin or neuraminidase. Additionally, the HA assay results showed a significant positive correlation with the infectious viral titer. Our results suggest that assessing the HA activity of PDCoV may be a useful diagnostic method for investigating and surveilling PDCoV infections.

Dynamics of pro- and anti-inflammatory cytokine changes in serum and assessment of their diagnostic utility during lactation impairment in pigs.

Postpartum dysgalactia syndrome (PDS) in sows is a frequent and important clinical problem in the field. Currently, the diagnosis is based on physical examination performed during first days after the farrowing. The present study aimed at evaluation the dynamics of pro- and anti-inflammatory cytokine (IL-4, IL-6, IL-8, IL-10 and TNF-α) changes in serum of sows during peripartum period (day - 28 to + 28) and assessment of their diagnostic utility during lactation impairment in pigs. The study was done on 139 sows divided into 3 groups: clinically healthy sows, sows with lactation disorders, sows which had experienced difficult parturitions, lameness, etc. In order to measure the level of serum cytokines, the quantitative species-specific ELISA assays were used. The investigation demonstrated a different kinetics of changes of studied cytokines in sows from various groups. IL-6 and TNF-alfa shown high dynamic changes after farrowing in in sows. The levels of IL-8 and IL-10 were relatively stable in healthy sows, while in sows with peripartum disorders usually increased during lactation. However, the detailed examination revealed that investigated cytokines cannot be a useful early diagnostic markers of lactation impairments in sow. They do not allow to detect with high probability which sows are susceptible to lactation disorders before the parturition.

Assessment of the safety and efficacy of an attenuated live vaccine based on highly virulent genotype 2b porcine epidemic diarrhea virus in nursing piglets.

We have previously reported the generation of the attenuated KNU-141112-S DEL5/ORF3 virus by continuous propagation of highly virulent G2b porcine epidemic diarrhea virus (PEDV) in Vero cells. The present study aimed to assess the safety of S DEL5/ORF3 and to evaluate its effectiveness as a live vaccine for prime-booster vaccinations. Reversion to virulence experiments revealed that the S DEL5/ORF3 strain retains its attenuated phenotype and genetic stability after five successive passages in susceptible piglets. Pregnant sows were primed orally with an S DEL5/ORF3 live vaccine and boosted intramuscularly twice with a commercial killed vaccine at 2-week intervals prior to parturition. This sow vaccination regimen completely protected nursing piglets against virulent G2b challenge, as evidenced by the increase in survival rate from 0% to 100% and the significant reduction in diarrhea intensity, including the amount and duration of PEDV fecal shedding. In addition, despite a 2-3 day period of weight loss in piglets from vaccinated sows after challenge, their daily weight gain was recovered at 7 days post-challenge and became similar to that of unchallenged pigs from unvaccinated sows over the course of the experiment. Furthermore, strong antibody responses to PEDV were verified in the sera and colostrum of immunized sows with the prime-boost treatment and their offspring. Altogether, our data demonstrated that the attenuated S DEL5/ORF3 strain guarantees the safety to host animals with no reversion to virulence and is suitable as an effective primary live vaccine providing durable maternal lactogenic immunity for passive piglet protection.

Antigenic and functional profiles of a Lawsonia intracellularis protein that shows a flagellin-like trait and its immuno-stimulatory assessment.

The obligate intracellular Lawsonia intracellularis (LI), the etiological agent of proliferative enteropathy (PE), is an economically important disease in the swine industry. Due to extreme difficulty of in vitro culture of the pathogen, molecular characterization of protein components of LI that are targets of the immune system, is difficult; thus, the scientific evidence to drive the development of preventive measures is lacking. In this work, we investigated the antigenic and functional characteristics of a putative flagellar-associated protein, LI0570, using in silico computational approaches for epitope prediction and an in vitro protein-based molecular assay. The amino acid sequence of LI0570 exhibited similarities to flagellar-associated proteins in four different bacterial strains. The presence of B cell linear confirmative epitopes of the protein predicted by a bioinformatics tool was validated by western blot analysis using anti-LI mouse hyperimmune serum, which implied that LI0570 induced production of antigen-specific antibodies in vivo. Further, TLR5-stimulating activity and IL-8 cytokine expression produced via downstream signaling were observed in HEK-Blue™-hTLR5 cells stimulated with LI0570. This result indicates that the LI0570 protein can trigger an innate immune response followed by a T-cell-related adaptive immune response in an infected host. Collectively, the data presented here support that the LI0570 protein which shows the antigenic potential could be a useful component of a recombinant vaccine against PE, providing progress toward an effective prevention strategy.

Preliminary assessment on potentials of probiotic B. subtilis RX7 and B. methylotrophicus C14 strains as an immune modulator in Salmonella-challenged weaned pigs.

A total of 40 crossbred weaned piglets (28 days old; [Landrace × Yorkshire] × Duroc) were used for preliminary assessment on potentials of Bacillus-based probiotics as an immune modulator in a Salmonella Typhimurium challenge model in a 3-week experiment. Pigs were randomly allotted to four experimental diets according to their initial body weight (9.21 ± 1.1 kg) and sex (10 pigs per treatment; 5 barrows and 5 gilts). The dietary treatments were basal diet (CON), basal diet + oral administration of Salmonella enterica ser. Typhimurium at the dosage of 1 mL containing 1 × 1011 cfu/mL of viable cell concentrations at day 21 (SC), SC + Bacillus subtilis (BS), and SC+ Bacillus methylotrophicus (BM). After 12 h of Salmonella challenge, the red blood cell (RBC), immunoglobulin G (IgG), and immunoglobulin M (IgM) concentrations were reduced (P < 0.05) whereas haptoglobin and cortisol levels were greater (P < 0.05) in SC compared with CON. However, the concentrations of RBC, IgG, and IgM were increased whereas haptoglobin and cortisol levels were reduced in BS and BM compared with SC. The probiotic-treated groups showed reduced (P < 0.05) IgM levels and increased (P < 0.05) WBC and cortisol levels compared with CON. The supplementation of probiotics showed increased (P < 0.05) fecal Lactobacillus counts and reduced Escherichia coli and Salmonella counts in piglets though there was no biological relevance compared with SC. Thus, in our preliminary study, Bacillus-based probiotic has shown some positive immunomodulatory effects in Salmonella-challenged pigs which provided a base for further studies.

Assessment of neutralizing and non-neutralizing antibody responses against Porcine circovirus 2 in vaccinated and non-vaccinated farmed pigs.

Vaccination is the most efficacious procedure to curtail Porcine circovirus 2 (PCV2)-associated diseases (PCVAD). Experimental studies indicate that PCV2 vaccine-induced virus-neutralizing antibodies play a major role in protection from PCVAD. However, the immune response to PCV2 vaccination of pigs on farms is less clear. Analysing groups of age-matched vaccinated and non-vaccinated farmed pigs, we found significantly increased levels of virus-neutralizing antibodies only in vaccinated pigs belonging to the age group with the highest risk for developing PCVAD. Serum levels of PCV2 genomes were not different between corresponding age groups. Levels of antibodies directed against a linear peptide from the PCV2 capsid protein correlated with those of virus-neutralizing antibodies and reached the highest levels in older, non-vaccinated animals, pointing towards an intense interaction between PCV2-infected cells and the immune system. In conclusion, current PCV2 vaccines are in need of improvement to induce stronger and more rapid immunity to prevent PCV2 infection.

Quantitative immunohistochemical assessment of IgA, IgM, IgG and antigen-specific immunoglobulin secreting plasma cells in pig small intestinal lamina propria.

Intestinal immune response plays an important defensive role for pathogens, particularly for those transmitted by the oro-faecal route or for foecal shedding modulation. This work examined three parts of intestine from twelve gilts experimentally infected with PCV2-spiked semen, six vaccinated (V group) and six unvaccinated (NV group) against PCV2, 29 and 53 days post infection (DPI). An immunohistochemical investigation for IgA-, IgG- and IgM-antibody bearing plasma cells (PCs) was run on intestinal samples coupled with a sandwich immunohistochemical method to reveal anti-PCV2 antibody-secreting PCs. Plasma cell density was compared in the two groups of animals at 29 and 53 DPI. The IgA, IgG and IgM PC density did not differ between groups but displayed an increase from the upper (villus) to the lower part of the crypts while a decreasing trend in PC density was identified from duodenum to ileum. In the NV group, no increase in anti-PCV2 PC density was demonstrable in the two sampling moment: the amounts of lamina propria PCV2-specific antibody-producing PCs remained constant, 10.55 ± 4.24 and 10.06 ± 5.01 at 29 DPI and 53 DPI, respectively. In the V group a significant increase in PCV2-specific antibody-producing PCs was observed over time. The amounts of PCV2-specific antibody-producing PCs increased from 9.37 ± 13.36 at 29 DPI to 18.76 ± 15.83 at 53 DPI. The data on IgA, IgM and IgG PC counts can be considered reference values in a population of adult pigs. The sandwich method can be proposed as a technique able to identify specific antibody-secreting PCs in formalin-fixed paraffin-embedded tissues. A practical application of the sandwich method is the demonstration of a "booster-like" response of the lamina propria in vaccinated compared to unvaccinated animals. After virus challenge, vaccination induced an increase in the number of PCs containing specific anti-PCV2 antibodies at the level of intestinal mucosa.

Assessment of an oral Mycobacterium bovis BCG vaccine and an inactivated M. bovis preparation for wild boar in terms of adverse reactions, vaccine strain survival, and uptake by nontarget species.

Wildlife vaccination is increasingly being considered as an option for tuberculosis control. We combined data from laboratory trials and an ongoing field trial to assess the risk of an oral Mycobacterium bovis BCG vaccine and a prototype heat-inactivated Mycobacterium bovis preparation for Eurasian wild boar (Sus scrofa). We studied adverse reactions, BCG survival, BCG excretion, and bait uptake by nontarget species. No adverse reactions were observed after administration of BCG (n = 27) or inactivated M. bovis (n = 21). BCG was not found at necropsy (175 to 300 days postvaccination [n = 27]). No BCG excretion was detected in fecal samples (n = 162) or in urine or nasal, oral, or fecal swab samples at 258 days postvaccination (n = 29). In the field, we found no evidence of loss of BCG viability in baits collected after 36 h (temperature range, 11°C to 41°C). Camera trapping showed that wild boar (39%) and birds (56%) were the most frequent visitors to bait stations (selective feeders). Wild boar activity patterns were nocturnal, while diurnal activities were recorded for all bird species. We found large proportions of chewed capsules (29%) (likely ingestion of the vaccine) and lost baits (39%) (presumably consumed), and the proportion of chewed capsules showed a positive correlation with the presence of wild boar. Both results suggest proper bait consumption (68%). These results indicate that BCG vaccination in wild boar is safe and that, while bait consumption by other species is possible, this can be minimized by using selective cages and strict timing of bait deployment.

Bayesian modelling of hunting data may improve the understanding of host-parasite systems: wild boar diseases and vaccination as an example.

Wildlife diseases are often studied using hunting data. In such studies, inferences about diseases are often made by comparing raw disease prevalence levels, ignoring complications like stochasticity in recruitment. We carried out a field trial to study the effectiveness of oral vaccination of wild boar (Sus scrofa) against classical swine fever (CSF) in the Vosges mountains (Northeastern France) for 3 years (2008-2010). Since August 2004, hunters had carried out three vaccination sessions per year in spring, summer and autumn. During our study period, we determined whether each wild boar hunted in our study area was immunized or not against CSF. We used a Bayesian approach to model the changes in the proportion of vaccinated animals in the population of young animals (i.e., <12 months old). This approach allowed to disentangle the effects of the birth peaks (leading to a decrease) and of both the vaccination sessions and natural infection (leading to an increase) on this proportion. We thus inferred, at the individual level, the probability that a non-immunized animal became vaccinated after a particular session. There was a high between-year variability in the effectiveness of the vaccination: the observed patterns were similar in 2008 and 2010, but 2009 was characterized by an overall greater effectiveness of the vaccination. Within a particular year, the spring vaccination session was more effective than the autumn session, probably because of the higher food availability in autumn that render the vaccination places less attractive to the animals. The vaccination effectiveness was rather low in summer, except in 2009, probably because of higher age identification error this year. This model also highlighted an immunisation of animals occurring outside vaccination periods, which suggests either the presence of the CSF virus in our study area, or the consumption of the vaccine outside the vaccination sessions. Finally, we observed a high spatial variability of the probability of vaccination. The effectiveness of the vaccination was indeed strongly related to both the distribution of the forests and the distribution of the vaccination places in our study area. This study highlights an optimal vaccination effort of 1.25 places per km(2) to maximize the proportion of immune wild boar in that area.

Development of the S3Pvac vaccine against murine Taenia crassiceps cysticercosis: a historical review.

Our work of the last 25 yr was concerned with the development of a vaccine aimed to prevent porcine Taenia solium cysticercosis and was based on cross-reacting Taenia crassiceps antigens that had proved protective against experimental intraperitoneal murine T. crassiceps cysticercosis (EIMTcC). In recent times the efficacy of the vaccine has been considered in need of confirmation, and the use of EIMTcC has been questioned as a valid tool in screening for vaccine candidates among the many antigens possibly involved. A review of our work divided in 2 parts is presented at this point, the first dealing with EIMTcC and the second with porcine T. solium cysticercosis (presented in this issue). Herein, we revise our results using EIMTcC as a measure of the protective capacity of T. crassiceps complex antigen mixtures, of purified native antigens, and of S3Pvac anti-cysticercosis vaccine composed by 3 protective peptides: GK-1, KETc1, and KETc12 either synthetic or recombinantly expressed and collectively or separately, by diverse delivery systems when administered at different doses and by different routes. Statistical analyses of the data lead confidently to the strong inference that S3Pvac is indeed an effective vaccine against EIMTcC via specific and non-specific mechanisms of protection.

Preliminary assessment of usefulness of cELISA test for screening pig and cattle populations for presence of antibodies against Toxoplasma gondii.

Serology testing is an appropriate method for the detection of slaughter animals infected with Toxoplasma, which remain one of the main reservoirs of this parasite in the environment. Competitive ELISA (cELISA) in indirect modification was worked out and optimized for detecting antibodies to Toxoplasma gondii in pigs and cattle. Preliminary validation process showed that the sensitivity and specificity of cELISA obtained in pigs was better than in cattle (88.1% and 94.5% vs. 76.9% and 93.4%, respectively). Sera of 861 pigs and 865 cattle were examined with newly worked out cELISA test and modified agglutination test (MAT) (Toxo-Screen DA, bioMérieux, France). In the total of 1,726 examined animal sera, seropositive results were obtained in 15.0% by cELISA (15.4% in pigs and 14.6% in catttle), and in 13.6% by MAT (14.3% in pigs and 12.8% in cattle). Significant differences in percentages of positive results among populations of the studied animals from various areas of Poland were noted. Obtained results showed the usefulness of cELISA for examining sera of slaughter animals (especially pigs). The considerable rates of infection of slaughter animals with T. gondii in the area of research indicate a potential threat to human health.

Improvement of the synthetic tri-peptide vaccine (S3Pvac) against porcine Taenia solium cysticercosis in search of a more effective, inexpensive and manageable vaccine.

Vaccination of pigs may curtail Taenia solium transmission by reducing the number of cysticerci, the precursors of adult intestinal tapeworms in humans. Several antigen preparations induce protection against porcine cysticercosis in experimental settings but only one subunit vaccine (S3Pvac) has been tested and proved effective in the field against naturally acquired disease. Besides improving of the vaccine's effectiveness, significant reductions in production costs and in the logistics of its administration are necessary for the feasibility of nationwide control programs. This review highlights the development of several versions of S3Pvac aimed to increase effectiveness, reduce costs and increase feasibility by novel delivery systems and alternative routes of administration.

Risk assessment of transmission of capsule-deficient, recombinant Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is the etiologic agent of swine pleuropneumonia. Live, non-encapsulated vaccine strains have been shown to be efficacious in preventing acute disease in pigs. Recombinant DNA technology has the advantage of generating defined mutants that are safe, but maintain critical immunoprotective components. However, some recombinant strains have the disadvantage of containing antibiotic resistance genes that could be transferred to the animal's normal bacterial flora. Using DNA allelic exchange we have constructed attenuated, capsule-deficient mutants of A. pleuropneumoniae that contain a kanamycin resistance (Kn(R)) gene within the capsule locus of the genome. Following intranasal or intratracheal challenge of pigs the encapsulated parent strains colonized the challenge pigs, and were transmitted to contact pigs. In contrast, the capsule-deficient mutants were recovered only from the challenged pigs and not from contact pigs. Each kanamycin-resistant colony type recovered from the respiratory or gastrointestinal tracts of pigs challenged with the recombinant strain was screened with a probe specific for the Kn(R) gene. All probe-positive colonies were assayed for the specific Kn(R) gene by amplification of a 0.9 kb fragment of the antibiotic resistance gene by PCR. The 0.9 kb fragment was amplified from the recombinant A. pleuropneumoniae colonies, but not from any of the heterologous bacteria, indicating there was no evidence of transmission of the Kn(R) gene to resident bacteria. Following aerosol exposure of 276 pigs with recombinant, non-encapsulated A. pleuropneumoniae the recombinant bacteria were not recovered from any nasal swabs of 75 pigs tested or environmental samples 18 h after challenge. Statistical risk analysis, based on the number of kanamycin-resistant colonies screened, indicated that undetected transmission of the Kn(R) gene could still have occurred in at most 1.36% of kanamycin-resistant bacteria in contact with recombinant A. pleuropneumoniae. However, the overall risk of transmission to any resident bacteria was far lower. Our results indicate there was little risk of transmission of capsule-deficient, recombinant A. pleuropneumoniae or its Kn(R) gene to contact pigs or to the resident microflora.

Recombinant bacteriophage-based multiepitope vaccine against Taenia solium pig cysticercosis.

The aim of this study was to test the capacity of recombinant phages to deliver antigens for vaccination against porcine cysticercosis. Thus, three peptides (KETc1, KETc12, GK1) and a recombinant antigen KETc7, previously proven to induce high levels of protection against pig cysticercosis, were expressed on the surface of the M13 bacteriophage at multiple copies. The pool of these four recombinant phages induced high levels of protection against an experimental murine cysticercosis. The immunogenicity of the phage vaccine preparation was therefore, tested in pigs, the natural host of Taenia solium. Subcutaneous or oral vaccination with these phages induced antigen-specific cellular immune responses in pigs. Preliminary data also points to the protective capacity of this recombinant phage vaccine against pig cysticercosis. The immunogenicity of these recombinant phages, together with the low cost of their production, make them a realistic candidate to be tested in pigs as an anti-cysticercus phage vaccine for field trials. This is the first report describing the application of a filamentous bacteriophage as a vaccine in large animals such as pigs, the only intermediate hosts of T. solium, a parasite of major medical importance in developing countries. The potential application of phages as a modern platform for vaccines for human and animal diseases is discussed.

Benefit-cost analysis of vaccination and preemptive slaughter as a means of eradicating foot-and-mouth disease.

OBJECTIVE: To assess relative costs and benefits of vaccination and preemptive herd slaughter to control transmission of foot-and-mouth disease (FMD) virus (FMDV). SAMPLE POPULATION: 2,238 herds and 5 sale yards located in Fresno, Kings, and Tulare counties of California. PROCEDURE: Direct costs associated with indemnity, slaughter, cleaning and disinfecting livestock premises, and vaccination were compared for various eradication strategies. Additional cost, total program cost, net benefit, and benefit-cost value (B/C) for each supplemental strategy were estimated, based in part on results of published model simulations for FMD. Sensitivity analyses were conducted. RESULTS: Mean herd indemnity payments were estimated to be dollars 2.6 million and dollars 110,359 for dairy and nondairy herds, respectively. Cost to clean and disinfect livestock premises ranged from dollars 18,062 to dollars 60,205. Mean vaccination cost was dollars 2,960/herd. Total eradication cost ranged from dollars 61 million to dollars 551 million. All supplemental strategies involving use of vaccination were economically efficient (B/C range, 5.0 to 10.1) and feasible, whereas supplemental strategies involving use of slaughter programs were not economically efficient (B-C, 0.05 to 0.8) or feasible. CONCLUSIONS AND CLINICAL RELEVANCE: Vaccination with a highly efficacious vaccine may be a cost-effective strategy for control of FMD if vaccinated animals are not subsequently slaughtered and there is no future adverse economic impact, such as trade restrictions. Although less preferable than the baseline eradication program, selective slaughter of highest-risk herds was preferable to other preemptive slaughter strategies. However, indirect costs can be expected to contribute substantially more than direct costs to the total cost of eradication programs.

Assessment of replication and virulence of attenuated pseudorabies virus in swine.

A nonclinical study was conducted to characterize the replication behavior of a modified live gE-deleted pseudorabies virus (PRV MS+1) in swine and potential for reversion to virulence after animal passages. Two to 3 week-old weaned pigs, negative for PRV, were maintained in isolation and challenged by intranasal instillation. For the first passage, 6 pigs were given 1 mL of PRV MS+1 (10(7.3)TCID(50)/mL) and 2 were necropsied at 3, 4 and 5 days post-inoculation (PI). Brain and secondary lymphoid tissues were collected, homogenized and the supernatants individually pooled for virus isolation, and PRV was recovered from each sample. No clinical signs of PRV infection were observed, but each pig had a nasal swab suspect or positive for PRV. For the second passage, 5 pigs were given 1 mL of the homogenate of mixed tissues from 1 animal in the previous passage (PRV at 10(1.9) TCID(50)/mL). At 5 days PI, all pigs were necropsied, and PRV was not recovered from their tissue homogenates or nasal swabs, and no clinical signs were observed. During a second attempt at a second passage, tissue homogenates from all pigs in the first passage (PRV at approximately 10(1.7)TCID50(50)/mL) were pooled and used to inoculate 15 pigs with 2 mL for 3 consecutive days. Ten pigs were monitored for clinical signs and seroconversion through 21 days PI, and 5 pigs were necropsied at 5 days PI. No clinical signs or PRV antibodies were detected in the 10 monitored pigs, and no PRV was recovered from the homogenates or nasal swabs of the 5 necropsied pigs. Thus, no evidence of reversion to virulence was demonstrated in pigs given the attenuated PRV.