Failure In Pregnant Sows Research Articles

Pseudorabies Virus: From Pathogenesis to Prevention Strategies.

Pseudorabies (PR), also called Aujeszky’s disease (AD), is a highly infectious viral disease which is caused by pseudorabies virus (PRV). It has been nearly 200 years since the first PR case occurred. Currently, the virus can infect human beings and various mammals, including pigs, sheep, dogs, rabbits, rodents, cattle and cats, and among them, pigs are the only natural host of PRV infection. PRV is characterized by reproductive failure in pregnant sows, nervous disorders in newborn piglets, and respiratory distress in growing pigs, resulting in serious economic losses to the pig industry worldwide. Due to the extensive application of the attenuated vaccine containing the Bartha-K61 strain, PR was well controlled. With the variation of PRV strain, PR re-emerged and rapidly spread in some countries, especially China. Although researchers have been committed to the design of diagnostic methods and the development of vaccines in recent years, PR is still an important infectious disease and is widely prevalent in the global pig industry. In this review, we introduce the structural composition and life cycle of PRV virions and then discuss the latest findings on PRV pathogenesis, following the molecular characteristic of PRV and the summary of existing diagnosis methods. Subsequently, we also focus on the latest clinical progress in the prevention and control of PRV infection via the development of vaccines, traditional herbal medicines and novel small RNAs. Lastly, we provide an outlook on PRV eradication.

Porcine reproductive and respiratory syndrome virus induces tight junction barrier dysfunction and cell death in porcine glandular endometrial epithelial cells

Reproductive failure caused by porcine reproductive and respiratory syndrome virus (PRRSV) is characterized by embryonic death and weak-born piglets and is associated with placental cell apoptosis and impairment of endometrial integrity. Here, we aimed to determine whether endometrial epithelial barrier function and viability were altered following PRRSV type 1 or type 2 infection. PRRSV inoculation was examined at the apical or basolateral side of porcine glandular endometrial epithelial cell cultures isolated from 4- to 6-month-old PRRSV-free herd gilts (n = 7 pigs). On the apical side, four days postinfection (4 dpi) with type 2 PRRSV, transepithelial electrical resistance decreased by 31% ± 5%, and paracellular permeability to fluorescein isothiocyanate-dextran (4 kDa) increased by 10-fold as compared with the mock and type 1 infection. Real-time polymerase chain reaction results revealed that both PRRSV types upregulated the mRNA expression of the barrier builder tight junction protein (TJ) Cldn5, but downregulated pore-forming TJ Cldn7. Additionally, the expression of other TJ genes, i.e., Cldn3 and Cldn8, was differentially increased by PRRSV type 1 and that of zonula occludens-1 was increased by PRRSV type 2. MTT assays indicated an increase in porcine glandular endometrial epithelial cell culture at 2–6 dpi following type 2 infection. Analysis of apoptosis using Annexin/propidium iodide staining combined with flow cytometry showed that the percentage of viable cells decreased, accompanied by a significantly higher dead cell population following PRRSV type 2 infection at 2–4 dpi. PRRSV type 1 infection also induced dead cells (>4%) at 2 dpi; however, the cell population recovered at 4 dpi. In conclusion, PRRSV type 2 infection caused more severe TJ barrier dysfunction and reduced cell viability compared with PRRSV type 1 infection in the porcine endometrium. Impairment in the membrane integrity of the maternal glandular endometrium may be the underlying mechanism of PRRSV-induced reproductive failure in pregnant sows.

Large-scale manufacture of VP2 VLP vaccine against porcine parvovirus in Escherichia coli with high-density fermentation.

Porcine parvovirus (PPV) virus-like particles (VLPs) are a potential vaccine candidate for the prevention of parvovirus-induced reproductive failure in pregnant sows. Currently, the Escherichia coli (E. coli) expression system is the most cost-efficient to express recombinant proteins. To overcome the limitations of protein misfolding and to prepare soluble highly bioactive antigen and high yields of protein, we optimized the PPV-VP2 gene, subcloned it into pET24a, pET26b, pET28a, and pET30a, and transformed it into E. coli BL21(DE3)-Tf16 competent cells. The pET28a plasmid was selected for further manipulations because it expressed high levels of the bioactive PPV-VP2 protein. Under optimal high-density fermenting conditions in a 70-L fermenter, the total yield of wet weight E. coli cells was 124.86g/L, and PPV-VP2 protein was 2.5g/L. After large-scale purification with Triton X-114 two-phase extraction as well as activated carbon powder adsorption, hemagglutination (HA) titers in the purified PPV-VP2 protein reached 219 and endotoxin was reduced to 2500 EU/mL. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) results indicated that the purified PPV-VP2 protein self-assembled into VLPs. Immunogenicity assays in guinea pigs and pigs indicated that the ISA-201 VG adjuvanted PPV-VP2 VLP vaccine elicited hemagglutination inhibition (HI) and virus neutralization (VN) antibody titers comparable with PPV commercial inactivated vaccines, whereas viral loads in the spleen and liver of challenged guinea pigs were significantly reduced. In conclusion, our study provides a method for producing the PPV-VLP vaccine against PPV infection in E. coli and may offer a novel strategy for the soluble expression of other vaccine antigens.

Detection and molecular characterization of porcine parvovirus in fetal tissues from sows without reproductive failure in Argentina

Porcine parvovirus (PPV) is one of many pathogens responsible for reproductive failure in pregnant sows. Several studies have reported the appearance of new PPV strains that differ from previous isolates both genetically and antigenically. Thus, the protective effects of commercially inactivated vaccines could not be complete. In South America, the information about PPV is limited. Thus, the aim of the present study was to detect and characterize the PPV strains present in 131 mummies or stillbirths from normal deliveries in sows from a commercial swine farm of Argentina that uses the commercial vaccine. PCR results showed that 17/131 were positive to PPV. Ten of these viruses were isolated and sequenced. All viruses were related to the PPV1 sequence (NADL-2), maintaining the amino acid differences in positions 436 (S–P) and 565 (R–K). This study is the first to report the isolation of PPV in Argentina and the results suggest that PPV can cross the placenta even in vaccinated sows, thus affecting some of the fetuses and being able to cause fetal death in sows without reproductive failure. The results also suggest that vaccination only reduces clinical signs and reproductive disorders and may thus not be a perfect tool to manage PPV infection. This study provides information that needs to be studied in depth to improve strategies to prevent and control PPV infection in swine farms.

Global miRNA, lncRNA, and mRNA Transcriptome Profiling of Endometrial Epithelial Cells Reveals Genes Related to Porcine Reproductive Failure Caused by Porcine Reproductive and Respiratory Syndrome Virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) can cause respiratory disease and reproductive failure in pregnant pigs. Previous transcriptome analyses in susceptive cells have mainly concentrated on pulmonary alveolar macrophages (PAM) and Marc-145 cells, and on the respiratory system. Some studies reported that apoptosis of placental cells and pig endometrial epithelial cells (PECs) is an obvious sign linked to reproductive failure in pregnant sows, but the mechanism is still unknown. In this study, Sn-positive PECs were isolated and apoptosis rates were assessed by flow cytometry. PRRSV-infected PECs exhibited apoptosis, indicative of their susceptibility to PRRSV. Subsequently, the whole transcriptome was compared between mock- and PRRSV-infected PECs and 54 differentially expressed microRNAs (DEmiRNAs), 104 differentially expressed genes (DEGs), 22 differentially expressed lncRNAs (DElncRNAs), and 109 isoforms were obtained, which were mainly enriched in apoptosis, necroptosis, and p53 signal pathways. Integration analysis of DEmiRNA and DEG profiles revealed two microRNAs (ssc-miR-339-5p and ssc-miR-181d-5p) and five genes (SLA-DQB1, THBS1, SLC3A1, ZFP37, and LOC100517161) participating in the apoptosis signal, of which THBS1 and SLC3A1 were mainly linked to the p53 pathway. Integration analysis of DEGs with DElncRNA profiles identified genes involved in apoptosis signal pathway are regulated by LTCONS_00010766 and LTCONS_00045988. Pathway enrichment revealed that the phagosome and p53 pathways are the two main signals causing apoptosis of PECs, and functional analysis revealed a role of miR-339-5p in regulating apoptosis of PECs after PRRSV inoculation.

Intranasal immunization of pigs with porcine reproductive and respiratory syndrome virus-like particles plus 2\u2032, 3\u2032-cGAMP VacciGrade\u2122 adjuvant exacerbates viremia after virus challenge

BackgroundPorcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive failure in pregnant sows and acute respiratory disease in young pigs. It is a leading infectious agent of swine respiratory complex, which has significant negative economic impact on the swine industry. Commercial markets currently offer both live attenuated and killed vaccines; however, increasing controversy exists about their efficacy providing complete protection. Virus-like particles (VLPs) possess many desirable features of a potent vaccine candidate and have been proven to be highly immunogenic and protective against virus infections. Here we explored the efficacy of PRRSV VLPs together with the use of a novel 2′, 3′-cGAMP VacciGrade™ adjuvant.MethodsAnimals were immunized twice intranasally with phosphate buffered saline (PBS), PRRSV VLPs, or PRRSV VLPs plus 2′, 3′-cGAMP VacciGrade™ at 2 weeks apart. Animals were challenged with PRRSV-23983 at 2 weeks post the second immunization. PRRSV specific antibody response and cytokines were measured. Viremia, clinical signs, and histological lesions were evaluated.ResultsPRRSV N protein specific antibody was detected in all animals at day 10 after challenge, but no significant difference was observed among the vaccinated and control groups. Surprisingly, a significantly higher viremia was observed in the VLPs and VLPs plus the adjuvant groups compared to the control group. The increased viremia is correlated with a higher interferon-α induction in the serum of the VLPs and the VLPs plus the adjuvant groups.ConclusionsIntranasal immunizations of pigs with PRRSV VLPs and VLPs plus the 2′, 3′-cGAMP VacciGrade™ adjuvant exacerbates viremia. A higher level of interferon-α production, but not interferon-γ and IL-10, is correlated with enhanced virus replication. Overall, PRRSV VLPs and PRRSV VLPs plus the adjuvant fail to provide protection against PRRSV challenge. Different dose of VLPs and alternative route of vaccination such as intramuscular injection should be explored in the future studies to fully assess the feasibility of such a vaccine platform for PRRSV control and prevention.

Improvement of indirect enzyme-linked immunosorbent assay for detection of Japanese encephalitis virus antibodies in swine sera

Japanese encephalitis (JE) is an important zoonosis caused by the mosquito-transmitted JE virus (JEV), which is a causative agent of reproductive failure in pregnant sows. Detection of JEV antibodies in swine is performed by hemagglutination inhibition (HI), virus neutralization (VN), and the plaque reduction neutralization test (PRNT). The most stringent PRNT is the 90% endpoint PRNT (PRNT90). These conventional assays are difficult to carry out in diagnostic laboratories with insufficient instruments or cell culture systems. An alternative assay that is easily conducted and time efficient is required. In this study, we improved the indirect enzyme-linked immunosorbent assay (I-ELISA) with clarified antigen for the detection of JEV antibodies. The I-ELISA results obtained from 175 swine serum samples were compared with HI, VN, and PRNT90 results. The sensitivity of I-ELISA was 91.8%, 95.0%, and 94.7% compared with HI, VN, and PRNT90 results, respectively. The specificity of I-ELISA was 92.2%, 94.7%, and 94.7% compared with HI, VN, and PRNT90 results, respectively. Moreover, the I-ELISA results were significantly correlated with the HI (r = 0.93), VN (r = 0.95), and PRNT90 (r = 0.92) results. These results suggest that the improved I-ELISA is useful for serosurveillance of JEV in swine.

Porcine parvovirus capsid protein expressed in Escherichia coli self-assembles into virus-like particles with high immunogenicity in mice and guinea pigs

Porcine parvovirus (PPV) is a causative agent of reproductive failure in pregnant sows. Classical inactivated vaccine is extensively used to control PPV infection, but problems concerning safety, such as incomplete inactivation may occur. In this study, a novel subunit vaccine against PPV based on virus-like particles (VLPs) formed from the complete PPV VP2 protein expressed in a prokaryotic system with co-expressed chaperones is reported. The VLPs have a similar size, shape, and hemagglutination property to the PPV. Immunization with these VLPs stimulated the neutralization antibody and hemagglutination inhibition (HI) antibody responses in mice and guinea pigs. The lymphocyte proliferation response and cytokine secretion was also induced in immunized guinea pigs comparable to those immunized with PPV inactivated vaccine. In addition, immunization with VLPs also significantly reduced the PPV content in the spleen of guinea pigs 14 days after the challenge with intact virus. These studies suggest that PPV VLPs created as described here could be a potential candidate for vaccine development.

Isolation and molecular and phylogenetic analyses of encephalomyocarditis virus from wild boar in central China

Encephalomyocarditis virus (EMCV) can infect many host species and cause acute myocarditis and respiratory failure in piglets, reproductive failure in pregnant sows. In this study, an EMCV strain, designated JZ1202, was isolated from semi-captive wild boars that presented with acute myocarditis and sudden death in central China. It was identified by hemagglutination inhibition (HI) assay, reverse transcription polymerase chain reaction (RT-PCR) and genome sequencing. The subsequent results showed that the virus could produce a specific cytopathic effect on BHK cells and could cause clinical symptoms and pathological changes in mice. Complete genome sequencing and multiple sequence alignment indicated that JZ1202 strain was 81.3%–99.9% identical with other isolates worldwide. Phylogenetic analysis of the whole genome, ORF, VP3/VP1 and 3D genes using neighbor-joining method revealed that JZ1202 isolate was grouped into lineage 1. The results of this study confirmed that an EMCV strain JZ 1202 isolated from wild boar in central China was fatal to mice and provided new epidemiologic data on EMCV in China.

Isolation, molecular and phylogenetic analysis of encephalomyocarditis virus strain GS01 in China

Encephalomyocarditis virus (EMCV) is a small non-enveloped, single-stranded RNA virus. It can infect many host species and cause acute myocarditis and respiratory failure in piglets, reproductive failure in pregnant sows. Diseases caused by EMCV currently affect the swine industry worldwide. In this study, an EMCV strain was isolated from an aborted fetus in western China. It was identified by reverse-transcription polymerase chain reaction (RT-PCR) and genome sequencing. The subsequent results showed that the virus could produce a specific cytopathic effect on BHK-21 cells and could cause severe clinical symptoms and pathological changes in mice. Complete genome sequencing and multiple sequence alignment indicated that the GS01 strain was 79.9–99.9% identical with other isolates worldwide. Phylogenetic analysis showed that EMCV isolates fell into five clusters: lineage 1, 2, 3, 4, and 5 based on the nucleotide sequences of the entire ORF and VP3/VP1 junction, as well as 3D gene. GS01 isolate was grouped into lineage 1. The results of this study confirmed that an EMCV strain GS01 isolated from an aborted pig fetus in western China was fatal to mice and provided new epidemiologic data on EMCV in China.

Phylogenetic comparison of porcine circovirus type 2 (PCV2) and porcine reproductive respiratory syndrome virus (PRRSV) strains detected in domestic pigs until 2008 and in 2012 in Croatia.

BackgroundPorcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) have been present for the last 2 decades in Croatia, causing large economical losses in the pig production. The clinical features of the infections are mostly manifested by the development of respiratory problems, weight loss and poor growth performance, as well as reproductive failure in pregnant sows. Even though the infections are continuously recognized in some regions in Croatia, the heterogeneity of the detected viral strains from 2012 has not yet been investigated. The objective of this study was to compare virus strains of PCV2 and PRRSV detected until 2008 in Croatia with strains isolated in 2012 to gain a better epidemiological understanding of these two infections.ResultsPCV2 and PRRSV strains detected in 2012 in fattening pigs from regions where these two diseases have been previously described were compared to strains that have been detected in the same regions within the past two decades. The phylogenetic analysis revealed that the circulating PCV2 and PRRSV strains are distantly related to the previously described Croatian viral strains. However, when compared to known isolates from the GenBank a high genetic identity of PRRSV isolates with isolates from Hungary, Denmark and the Netherlands was found.ConclusionThe results of this study reveal that even though PCV2 and PRRSV are constantly present in the investigated regions in Croatia, the viral strains found in 2012 genetically differ from those detected in earlier years. This indicates that new entries into the pig population appeared with regard to both infections, probably as a result of pig trade.

Pathogenesis and prevention of placental and transplacental porcine reproductive and respiratory syndrome virus infection

Porcine reproductive and respiratory syndrome virus (PRRSV)-induced reproductive problems are characterized by embryonic death, late-term abortions, early farrowing and increase in number of dead and mummified fetuses, and weak-born piglets. The virus recovery from fetal tissues illustrates transplacental infection, but despite many studies on the subject, the means by which PRRSV spreads from mother to fetus and the exact pathophysiological basis of the virus-induced reproductive failure remain unexplained. Recent findings from our group indicate that the endometrium and placenta are involved in the PRRSV passage from mother to fetus and that virus replication in the endometrial/placental tissues can be the actual reason for fetal death. The main purpose of this review is to clarify the role that PRRSV replication and PRRSV-induced changes in the endometrium/placenta play in the pathogenesis of PRRSV-induced reproductive failure in pregnant sows. In addition, strategies to control placental and transplacental PRRSV infection are discussed.

A novel vaccine combined with an alum adjuvant for porcine encephalomyocarditis virus (EMCV)-induced reproductive failure in pregnant sows

To evaluate a novel vaccine for porcine encephalomyocarditis virus (EMCV), which causes reproductive failure in pregnant sows, virus like particles (VLPs) were generated and immunized twice in 2week intervals before sow mating. Sows were divided into 4 groups (n=4, per group). Group 1 was immunized with the alum adjuvant alone, Group 2 with VLPs alone, Group 3 with VLPs mixed alum adjuvant, and Group 4 with a commercial killed vaccine. In Group 2, seroconversion was observed at very low levels, while in Group 3, neutralizing antibodies were maintained at a high level until 30days after farrowing. Similar levels neutralizing antibodies were observed in Group 4. The gestation period of the pregnant sows was on average 115days, and no injection site reaction or side effects were observed. The mean temperature of the sows after immunization increased temporarily to 38.7–39.1°C for 1day. The numbers and weights of surviving piglets were similar among the groups. These data describe a novel EMCV vaccine composed of VLPs mixed with an alum adjuvant that is safe to use during sow gestation and induces and maintains high levels of seroconversion. This vaccine could thus be a candidate for protecting against EMCV induced reproductive failure in pig farms.

Development and evaluation of indirect ELISA for the detection of antibodies against Japanese encephalitis virus in swine

The Japanese encephalitis virus (JEV) is one of causative agents of reproductive failure in pregnant sows. An indirect enzyme-linked immunosorbent assay (I-ELISA) was examined for its potential use in the rapid monitoring of the JEV, and the results were compared with those from the hemagglutination inhibition (HI) and serum neutralization (SN) tests. The comparative analysis showed that the results of I-ELISA showed a significant correlation with the conventional HI (r = 0.867) and SN tests (r = 0.804), respectively. When the I-ELISA results were compared with the traditional diagnostic assays, the sensitivity of the I-ELISA was 94.3% with the HI test and 93.7% with the SN test, respectively. The specificity was found to be 81.4% and 80.0% with the HI and SN tests, respectively. To determine the applicability of I-ELISA in the field, the serum samples from 720 pigs were collected from 4 regions in Korea between July and August 2004. The results indicated that 21.7% of screened pigs were seropositive for the JEV. The seropositive rates of JEV in the 4 provinces were 12.6% in Gyeonggi, 45.0% in Gyeongnam, 16.7% in Jeonbuk, and 12.2% in Jeju. The I-ELISA methodology developed in this study was shown to have considerable sensitivity and specificity through a comparison with HI and the SN tests. Therefore, it might be one of convenient methods for screening a large number of samples in various fields.

The level of virus-specific T-cell and macrophage recruitment in porcine reproductive and respiratory syndrome virus infection in pigs is independent of virus load.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the most important infectious disease agent of pigs worldwide, causing reproductive failure in pregnant sows and respiratory problems in nursing and growing pigs. PRRSV infection is characterized by a prolonged viremia of 30 or more days and an extended persistent infection of lymphoid tissues. To better understand the immunological basis for prolonged acute and persistent PRRSV infection, we have examined the cell-mediated immune (CMI) response throughout the course of infection and compared the results to the local distribution and abundance of PRRSV in infected tissues. PRRSV-specific T cells, enumerated by gamma interferon enzyme-linked immunospot assay, did not appear until 2 weeks after PRRSV inoculation, and their abundance exhibited substantial variation over time and among animals. In all cases the T-cell response was transient. High levels of viral RNA were present in lymphoid tissues of all animals in the acute phase of infection. Viral loads were decreased 1,000-fold or more in persistent infections, with the primary sites of persistence being tonsil, sternal lymph node, and inguinal lymph node. The abundance of virus-specific T cells in either acutely or persistently infected animals was highly variable and showed no correlation to the level of virus in lymphoid tissues. No significant difference in antigen-specific T-cell abundance was observed in secondary lymphoid tissues in either acute or persistent infection except for tonsil, in which the number of responding cells was extremely low. CD4(+)- and CD8(+)-T-cell frequencies did not change after PRRSV infection, though a decrease in gammadelta T cells was observed. Macrophages, the permissive cell type for PRRSV, were present in various levels in all tissue preparations and were not in proportion to local virus load. These findings indicate that a weak CMI response contributes to prolonged PRRSV infection and suggests that PRRSV suppresses T-cell recognition of infected macrophages. Thus, the slow but eventual resolution of PRRSV infection may be dependent on limiting permissive macrophages and on innate immune factors.

Developmental toxicity of purified fumonisin B1 in pregnant Syrian hamsters.

The purpose of this study was to characterize the developmental toxicity of fumonisin B1 (FB1), a mycotoxin produced by Fusarium moniliforme, on fetal Syrian hamsters. Fusarium moniliforme has been associated with a variety of diseases in animals and esophageal cancer in humans. Purified FB1 causes leukoencephalomalacia in horses and is hepatocarcinogenic in rats. Fumonisin B1 has been associated with fetal toxicity in rats and mice and has been suggested to be involved in reproductive failure in pregnant sows. Results from a preliminary developmental toxicity study using an aqueous extract of F. moniliforme corn-culture material in hamsters suggested that FB1 was a developmental toxicant. These results were verified using purified FB1. Six groups of ten time-mated female Syrian hamsters were dosed with 0.0-18 mg kg(-1) day(-1) of FB1 by gavage on days 8-12 of gestation and euthanized on day 15. Live fetuses were weighed and examined for gross external and internal abnormalities and skeletal anomalies. Purified fumonisin B1 was shown to cause dose-dependent fetal death and delayed fetal development without causing fetal abnormalities.

Developmental toxicity of purified fumonisin B1 in pregnant Syrian hamsters

The purpose of this study was to characterize the developmental toxicity of fumonisin B1 (FB1), a mycotoxin produced by Fusarium moniliforme, on fetal Syrian hamsters. Fusarium moniliforme has been associated with a variety of diseases in animals and esophageal cancer in humans. Purified FB1 causes leukoencephalomalacia in horses and is hepatocarcinogenic in rats. Fumonisin B1 has been associated with fetal toxicity in rats and mice and has been suggested to be involved in reproductive failure in pregnant sows. Results from a preliminary developmental toxicity study using an aqueous extract of F. moniliforme corn-culture material in hamsters suggested that FB1 was a developmental toxicant. These results were verified using purified FB1. Six groups of ten time-mated female Syrian hamsters were dosed with 0.0–18 mg kg−1 day−1 of FB1 by gavage on days 8–12 of gestation and euthanized on day 15. Live fetuses were weighed and examined for gross external and internal abnormalities and skeletal anomalies. Purified fumonisin B1 was shown to cause dose-dependent fetal death and delayed fetal development without causing fetal abnormalities. © 1998 John Wiley & Sons, Ltd.

Efficacy of a mixed glycoprotein vaccine against pseudorabies in pregnant sows.

A pseudorabies virus (PRV) glycoprotein-mixed vaccine was prepared by heparin-affinity chromatography from PRV-infected PK-15 cell lysates. In our previous study, the trial vaccine was induced protection with suppression of virus shedding in one-month-old pigs and generation of cytotoxic T lymphocyte (CTL) response in mice. In this study, the effect of the trial vaccine on suppression of both virus shedding and reproductive failure in pregnant sows was examined. Three sows were vaccinated twice until one week before mating. Each of them was infected intranasally with 10(6) TCID50 of PRV on day 28, 54, or 85 after mating, respectively. Three other sows were also mated and challenged at the same time as the respective control. The vaccinated sows produced virus-neutralizing antibodies. Sows with high level of VN antibody lowered the level and period of virus shedding after challenge. The maximum level of shed-virus titers in vaccinated sows were 10(1.25) to 10(3) times lower than controls. Vaccinated sows shed virus for 1 or 5 days, while controls shed for 8, 9, or 12 days. No abortion or stillbirth was observed in vaccinated sows during pregnancy. On the other hand, control sow challenged at a late stage of pregnancy showed abortion and stillbirth. The results obtained here indicate that our trial vaccine is effective to prevent reproductive failure by pseudorabies virus.

Evaluation of an indirect fluorescent IgM antibody test for the detection of pigs with recent infection of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) has caused major economic losses to swine producers in recent years due to severe reproductive failure in pregnant sows and respiratory disease in young pigs. Antibodies to PRRSV in swine sera can be tested by various serologic methods including immunoperoxidase monolayer assay, 12 indirect fluorescent antibody (IFA) test, 14 enzymelinked immunosorbent assay, and serum neutralization (SN) test 3,15 However, none of the tests can adequately determine the time of infection. Development of a serologic test that could estimate the infection time or identify a recent infection would be valuable to swine farmers when introducing pigs from a PRRSV-infected farm. Demonstration of specific IgM antibodies has been a useful marker for recent infection to different viral infections. Virusspecific IgM antibody has been detected regularly in primary infection with herpes simplex virus, rubella virus, cytomegalovirus, hepatitis A and B viruses and pseudorabies viTUS. For example, IgM antibody to pseudorabies virus in pigs was first detected in serum 6 days postinoculation (PI), reached the highest titers 10 days PI, and was detectable up to 35 days PI. The objectives of this study were to evaluate an IFA test for the detection of IgM antibody specific to PRRSV and to compare the detection of IgM antibody and the presence of virus in sera from pigs with or without known days PI. PRRSV MN-1b was used for the IFA test, and the MN-H and MN-W isolates were used for animal inoculation. A permissive clone (MARC-145) of the African green monkey kidney (MA-104) cell line was used for virus propagation, virus isolation, and serology. The MARC-145 cells were maintained in Eagle’s minimum essential medium supplemented with 3% fetal bovine serum, 0.15% sodium bicarbonate, and antibiotics. Sera used were collected at known intervals from 3-weekold pigs following experimental infection with PRRSV MNH. Sera from sows infected experimentally with PRRSV MN-W or MN-H were also used. Field serum samples were collected from pigs of known age in 2 different farms with endemic PRRSV infection, and the samples were transported on the same day to the laboratory for virus isolation and serology. Virus isolation from sera was performed using 24-well microplates by inoculating 0.05 ml of the undiluted sera to 1 ml of MARC-145 cell suspensions (2 x 10 cell/ml). Infected monolayers were observed daily for cytopathic changes

Seroprevalence of indirect fluorescent antibody to porcine reproductive and respiratory syndrome virus in selected swine herds.

Porcine reproductive and respiratory syndrome (PRRS) virus has recently been identified in the USA as a cause for reproductive failure in pregnant sows and respiratory disease in young pigs. 1-4,6 The syndrome has been commonly referred to as swine infertility and respiratory syndrome in this country but there was a general agreement to adopt the name PRRS at an international PRRS symposium (St. Paul, MN, May 1992). A virus designated as Lelystad virus has also been reported to cause a similar syndrome in several European countries. 1,5 Both viruses can cause significant economic problems for swine producers because of acute reproductive losses and piglet mortality. Chronic production losses involving increased mortality and reduced growth rate in pigs at postweaning age have also been reported on some infected farms. 1 In our recent study, an indirect fluorescent antibody (IFA) test was developed to detect and quantitate PRRS virus antibody in swine sera. 7 This IFA test for swine sera collected between 1981 and 1991 revealed evidence of PRRS virus infection in this country as early as April 1986. The purpose of the present study was to report the recent Seroprevalence status of IFA to PRRS virus by testing sera collected from selected swine herds during the first 6 months of 1992. Swine sera submitted January-June 1992 for serodiagnosis to the Oxford Veterinary Laboratory, Worthington, Minnesota, were used. Since October 1988, the laboratory has provided a swine antibody profile service to practicing veterinarians for different pathogens. The service has included serology for Actinonobacillus pleuropneumonia serotypes 1, 5, and 7, encephalomyocarditis virus, porcine parvovirus, Leptospira spp., and group D Streptococcus. The IFA test for PRRS virus was included starting in January 1992. Between January and June 1992, a total of 2,787 sera from 263 swine farms (an average of 10.6 serum samples per farm), were submitted from 13 different states. Veterinarians provided a brief farm history and requested serology for specific agents. Of the 263 farms, 225 requested PRRS virus serology. For the PRRS virus IFA test, aliquots of each sample were sent to the swine virology laboratory, University of Minnesota. ethanol was used to fix the cell monolayers in each well. Test sera, diluted 4-fold serially from 1:4 to 1:1,024, were added to test plates. The plates were incubated, washed, and incubated again with an optimal dilution of rabbit anti-swine IgG conjugated with fluorescein isothiocyanate. The plates were then washed and examined under a fluorescent microscope. Negative and positive reference sera and an uninfected SAM control for each serum were included in each test. The IFA titers were recorded as the highest serum dilutions with specific cytoplasmic fluorescence, and titers of ≤ 1:4 and ≥ 1: 16 were considered negative and positive, respectively. Of 2,787 sera from 263 swine herds tested, 979 sera (35.1%) had PRRS IFA titers of 21: 16 and 148 farms (56.3%) had 1 or more pigs that were PRRS virus IFA test positive (Table 1). Of 1,726 sera from 148 farms with PRRS virus seropositive pigs, 747 sera (43.3%) had IFA titers of ≤ 1:4 and 979 sera (56.7%) had the titers of ≥ 1:16. Of 979 seropositive samples, 222 (22.7%) 314 (32.1%), 283 (28.9%), and 160 (16.3%) samples had IFA titers of 1:16, 1:64, 1:256, and ≥ 1: 1,024, respectively. Of 225 farms requesting PRRS serology, 129 (57.3%) had seropositive pigs, whereas 19 of 38 farms (50.0%) that did not request PRRS virus serology were positive. Results of the farms with seropositive pigs were 1 1/16 (68.8%), 16/41 (39.0%) 35/65 (53.8%) 21/35 (60.0%), 37/61 (60.7%), and 28/45 (62.2%) farms for January, February, March, April, May, and June, respectively. No significant difference for seropositive farms was found between the months by chisquare analysis (P = 0.190). The positive farms were located in 11 different states.