J. M. Castro

Analysis of cellular immune response in pigs recovered from porcine respiratory and reproductive syndrome infection

The cellular immune response to a European isolate of porcine reproductive and respiratory syndrome (PRRS) virus in animals recovered from the experimental infection has been studied in vitro. Peripheral blood mononuclear cells (PBMC) from these pigs proliferated specifically when they were stimulated with PRRS virus. This response was not detectable until 4 weeks after inoculation and remained for more than 3 months. Addition of blocking monoclonal antibodies to the cultures showed that this proliferation was mainly dependent on CD4(+) cells with the participation of SLA-class II molecules. T-cell cultures established by stimulating responding cells with PRRS virus and maintained in culture for up to 3 weeks showed an increase of CD8(+) CD4(+) and CD4(-) CD8(+) subsets within activated cells, gated according to their light scatter parameters, whereas CD4(+) CD8(-) cells declined along the time in culture. Within the activated cells, those expressing the TcR gammadelta receptor also increased, being most of them also positive for the CD8 marker. By RT-PCR, T-cells responding to the virus showed a Th1 type cytokine production pattern. During the culture period the cytotoxic activity against K-562 cells increased from 15 to 35% of specific lysis. This cellular immune response may play a relevant role in the clearance of PRRS virus and the recovery of the infection.

Phylogenetic relationships of European strains of porcine reproductive and respiratory syndrome virus (PRRSV) inferred from DNA sequences of putative ORF-5 and ORF-7 genes

The complete ORF-5 gene and a fragment of the ORF-7 gene from 14 different European porcine reproductive and respiratory syndrome virus (PRRSV) isolates were amplified by reverse transcriptase polymerase chain reaction (RT-PCR), and their nucleotide sequences were determined. The ORF-7 gene displayed nucleotide and amino acid identities of 94.1-99.6% and 95.3-100% among isolates from different countries. The ORF-5 gene showed higher nucleotide (87.1-99.2% identity) and amino acid (-88% identity) variability. The resulting sequences were aligned with other European and North American PRRSV strains and phylogenetic relationships among these strains were established by the maximum parsimony method. The phylogenetic trees inferred from both genes were in agreement and showed that European and North American PRRSV strains clearly represent two different genotypes. According to both trees, there is a perfect correlation between strains and the countries in which they were isolated. Additionally, the phylogenetic position of European and North American PRRSV strains within the recently proposed family Arteriviridae was also analyzed.

Direct detection of the porcine reproductive and respiratory syndrome (PRRS) virus by reverse polymerase chain reaction (RT-PCR)

SummaryA method for direct detection of the porcine reproductive and respiratory syndrome (PRRS) virus was developed, based on reverse transcription of the viral RNA coupled to DNA amplification by polymerase chain reaction. A set of primers was designed from Lelystad virus sequence within ORF 7 encoding nucleocapsid protein. From seven Spanish field isolated strains the 312 bp amplified fragment was cloned and sequenced. Alignment with Lelystad virus sequence revealed a 96–97% homology. A maximum sensitivity of 6.7 TCID50 was achieved with the reported procedure in experimentally infected swine alveolar macrophages cultures. The sensitivity obtained in crude clinical samples from experimentally infected 3-weeks old pigs was approximately 102 TCID50. High specificity for the PRRS virus was demonstrated for the method, as none of the seven common swine virus assayed rendered DNA amplification product.

Porcine reproductive and respiratory syndrome (PRRS) virus down-modulates TNF-α production in infected macrophages

The effect of porcine reproductive and respiratory syndrome (PRRS) virus infection on the synthesis and secretion of TNF-alpha and other pro-inflammatory cytokines by porcine alveolar macrophages (PAM) was investigated as well as the effect that TNF-alpha has on the replication of this virus. A clear reduction of phorbol myristate acetate (PMA)-induced expression of TNF-alpha mRNA was observed in cells incubated with PRRS virus. Moreover, the presence of PRRS virus also induced a decrease in IL-1 alpha and MIP-1 beta mRNAs expression with respect to PMA-stimulated uninfected cells. According to these results, exposure to the PRRS virus led to a reduction of the TNF-alpha protein in supernatants of PMA-stimulated PAM. On the other hand, addition of recombinant porcine TNF-alpha to cultures clearly reduced virus replication; however the addition of TNF-alpha to cultures containing IFN-alpha did not result in a further reduction of the produced by IFN-alpha alone. This indicates the lack of synergy in the effect of these cytokines on viral replication.

Failure of an inactivated vaccine against porcine reproductive and respiratory syndrome to protect gilts against a heterologous challenge with PRRSV.

This study was designed to evaluate the efficacy of an inactivated vaccine based on a European-type strain of porcine reproductive and respiratory syndrome virus (prrsv) against the reproductive form of the syndrome in breeding gilts, and any congenital disease in their piglets. Five gilts were vaccinated twice, following the manufacturers instructions, before they were inseminated. Nine additional gilts remained unvaccinated and served as positive (five gilts) and negative (four gilts) controls. A European wild-type strain genetically divergent from the vaccine strain was used to challenge the five vaccinated and five unvaccinated positive control gilts at 90 days gestation. The vaccination of the five seronegative gilts did not produce any clinical signs or adverse reactions. However, the vaccine failed to prevent the clinical signs associated with prrsv infection, viraemia after the challenge and transplacental infection of their piglets. The reproductive performance of the vaccinated gilts was similar to that of the unvaccinated positive controls, and there were no statistically significant differences in most of the parameters tested. However, the preweaning mortality of the piglets born to the vaccinated gilts was significantly lower than that of the piglets born to the positive control gilts.

Rapid and high sensitivity test for direct detection of bovine herpesvirus — 1 genome in clinical samples

A procedure for direct detection of the BHV-1 genome in clinical samples, including semen, was developed. The method is based on the PCR amplification of a highly conserved DNA fragment within the glycoprotein gI sequence of the virus (323 bp between nt 1491 to nt 1814). The method is rapid and highly specific for all 27 subtypes assayed, which are included in the clinical and genetically different groups of BHV-1. The viral origin of the PCR product was assessed by Southern hybridization, with an internal probe. The method for DNA isolation from clinical samples included a fast extraction procedure with Chelex 100 resin allowing the loading of larger amounts of DNA in the PCR and in turn increasing the sensitivity of the method of detection. The level of sensitivity achieved by PCR was in the range of 1 TCID(50). This PCR assay may be an useful tool for BHV-1 monitoring in semen banks at low cost.

Insemination of susceptible and preimmunized gilts with boar semen containing porcine reproductive and respiratory syndrome virus

Twenty-one gilts without measurable PRRSV serum antibody titres were identified for this experiment. Seven gilts were used as controls (Group C) and 14 as principals. Of these, 7 gilts were preimmunized to PRRSV and constituted Group B, while 7 gilts remained seronegative and constituted Group A. The principal gilts were inseminated with boar semen containing PRRSV and were killed 20 d later. The control gilts were treated similarly but were not exposed to PRRSV. Gilts were observed for clinical signs of infection. The effects on the conception rates were studied and gilts and embryos were tested for PRRSV and homologous antibodies. Group A and B gilts developed signs of PRRS associated with anorexia and slightly elevated body temperatures. Transmission of the infection was demonstrated by the isolation of PRRSV from serum and other tissue samples of principal gilts and also by seroconversion. The results show that early infection may have an insignificant effect or no effect on the conception and fertilization rates. However, exposure to PRRSV at the time of insemination can result in transplacental infection of embryos. In Group A gilts, 5 of 6 litters were infected prenatally with 7.6% of embryos infected. In Group B gilts, 1 of 5 litters and 1.3% of embryos were infected. Moreover, approximately 2 and 4 times more embryos were dead in litters of gilts from Group A and Group B than in gilts from control Group C. The isolation of PRRSV in 3 dead embryos suggests that the embryos may have died as a result of the direct effect of the virus. It can be concluded that the insemination of either seronegative or preimmunized gilts with boar semen containing PRRS V may have an insignificant effect or no effect on conception and fertilization rates, although it can result in transmission of the virus and embryonic infection and death.

Semen changes in boars after experimental infection with porcine reproductive and respiratory syndrome (PRRS) virus

Eleven boars seronegative to porcine reproductive and respiratory syndrome virus (PRRSV) were trained for semen collection: five boars were inoculated intranasally with 6 x 10(6)TCID(50)/ml of PRRSV (Group A); four boars were inoculated intranasally with 6 x 10(4)TCID(50)/ml (Group B); and two boars were used as uninfected control (Group C). Semen samples were collected at 7-d intervals from 49 d prior to experimental inoculation with PRRSV to 70 d after inoculation, and were examined for sperm volume, sperm concentration, sperm morphology, sperm motility and for the presence of PRRSV. The infection in boars was demonstrated by the reisolation of PRRSV from the serum of all inoculated boars. Rectal temperatures and general health of the boars were clinically normal throughout the trial. Differences were observed in the quality of semen collected from boars after experimental infection with PRRSV. This infection induced a significant decrease in sperm motility and in spermatozoa with normal acrosomes. Of the semen samples tested for virus isolation in swine alveolar macrophages PRRSV was only isolated in 1 boar from Group B. The virus was detected in an additional semen sample in Group A by the production of an antibody titer in a biological assay. All attempts to detect PRRSV by RT-PCR in semen samples were unsuccessful. Nevertheless, from our study it is possible to suggest that the PRRSV can occasionally be transmitted in the semen during the initial phase of the disease.

Transplacental infection following exposure of gilts to porcine reproductive and respiratory syndrome virus at the onset of gestation

Twenty-five gilts without measurable porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) serum antibody titres were used for this experiment. All of them were randomly assigned to one of the treatment groups at the time of artificial insemination. Twelve gilts were exposed to PRRSV, of these, six were slaughtered on day 10 after exposure and constituted group A. The remaining six were slaughtered on day 20 after infection and constituted group C. Thirteen gilts were used as controls, six of these were slaughtered on day 10 after treatment and constituted group B. The remaining seven were slaughtered on day 20 after treatment and constituted group D. The infected gilts were inoculated with PRRSV intranasally and intravenously in the ear vein. They were observed for clinical signs of infection and the effects on conception and fertilization rates were studied, while the gilts and their embryos were tested for PRRSV and homologous antibodies. The infected animals developed signs of PRRS associated with anorexia and slight pyrexia. Infection was verified by reisolation of the virus from serum and other tissue samples and also by seroconversion. Ten out of 12 infected gilts and 10 out of 13 controls were pregnant at the time of slaughter and the ratio of embryos to corpora lutea was the same in both, infected and control groups (0.75). Therefore, infection with PRRSV at the onset of gestation did not appear to interfere with conception and fertilization rates and subsequent pregnancy. The PRRSV was not isolated from any of the embryos collected at day 10 postexposure, but was present in 20-day-old embryos of group C gilts. In this group, 60% of litters were infected prenatally, with 16% of embryos infected. The proportion of dead embryos was three times greater than in a control group D (35.4% and 9.8%, respectively). The results of this report indicate that exposure of susceptible gilts to PRRSV at the onset of gestation has no significant effect on conception and fertilization rates. However, although infection does not appear to have any effect on the embryos before implantation, it can result in transplacental infection and embryo death.

Exposure of gilts in early gestation to porcine reproductive and respiratory syndrome virus

Twenty-five gilts without measurable serum antibody titres to porcine reproductive and respiratory syndrome virus (PRRsv) were identified and 16 were inoculated with PRRSV at seven, 14 or 21 days of gestation and killed 20 to 22 days later to determine the effect of the virus on their embryos. The remaining nine gilts were not exposed to PRRSV, but were killed at the same stages of gestation. The gults were observed for clinical signs of infection and the gilts and their embryos were tested for PRRv and homologous antibodies. The infection was demonstrated by the re-isolation of the virus and its detection by the reverse transcriptase polymerase chain reaction in serum and other tissue samples from the inoculated gilts, and also by seroconversion. However, the gilts remained healthy throughout the study, except for one which was depressed and anorexic for two days. Two of the litters from the gilts challenged with PRRSV on day 14 of gestation contained one and three infected live embryos; the other embryos from these two litters did not contain detectable virus, although most of the embryos in one of the litters were dead. The other nine litters from the gilts challenged with PRRSV and the control litters, showed no evidence of infection.