Immunostimulatory effects of recombinant Erysipelothrix rhusiopathiae expressing porcine interleukin-18 in mice and pigs.

Interleukin-18 (IL-18), which was originally called gamma interferon (IFN-γ)-inducing factor, has been shown to play an important role in innate and acquired immune responses. In this study, attenuated Erysipelothrix rhusiopathiae strains were engineered to produce porcine IL-18 (poIL-18) and evaluated for their potential immunostimulatory effect in animals. Recombinant poIL-18 was successfully expressed in the recombinant E. rhusiopathiae strains YS-1/IL-18 and KO/IL-18. The culture supernatant of YS-1/IL-18 was confirmed to induce IFN-γ production in murine splenocytes in vitro, and this production was inhibited by incubation with anti-poIL-18 monoclonal antibodies. Furthermore, more IFN-γ production was induced upon stimulation of splenocytes with concanavalin A for splenocytes from mice that were intraperitoneally inoculated with YS-1/IL-18 than for splenocytes from control mice inoculated with the parent strain YS-1. Peritoneal macrophages from mice preinoculated with YS-1/IL-18 exhibited enhanced phagocytosis of Salmonella enterica subsp. enterica serovar Typhimurium compared with peritoneal macrophages from control mice preinoculated with YS-1. We also confirmed the immunostimulatory effect on humoral immune responses against antigens of E. rhusiopathiae and Mycoplasma hyopneumoniae in gnotobiotic pigs that were orally preinoculated with KO/IL-18. Thus, these results provide evidence that E. rhusiopathiae is a promising vector for the expression of host cytokines and suggest the potential utility of E. rhusiopathiae vector-encoded cytokines in the activation of host innate and acquired immune responses.

Allele-specific primer polymerase chain reaction for a single nucleotide polymorphism (C1205T) of swine toll-like receptor 5 and comparison of the allelic frequency among several pig breeds in Japan and the Czech Republic.

In the present study, an allele-specific primer-polymerase chain reaction (ASP-PCR) for genotyping a single nucleotide polymorphism (SNP) of swine Toll-like receptor 5 (TLR5) (C1205T; P402L) that is related to the impaired recognition of Salmonella enterica serovar Choleraesuis (SC) was developed. The allele frequencies in several pig breeds in Japan and the Czech Republic were also compared. The swine TLR5 C1205T mutation was successfully determined by ASP-PCR using genomic DNA samples in Japan that had previously been genotyped by a sequencing method. Using the PCR condition determined, genomic DNA samples from blood obtained from 110 pigs from seven different breeds in the Czech Republic were genotyped by the ASP-PCR. The genotyping results from the ASP-PCR completely matched the results from the sequencing method. The allele frequency of the swine TLR5 C1205T mutation was 27.5% in the Landrace breed of the Czech Republic compared with 50.0% in Japanese Landrace. In Japan, the C1205T mutation was found only in the Landrace breed, whereas in the Czech Republic it was found in both the Landrace and Piétrain breeds. These results indicate the usefulness of ASP-PCR for detecting a specific SNP for swine TLR5 affecting ligand recognition. They also suggest the possibility of genetically improving pigs to enhance their resistance against SC infection by eliminating or selecting this specific SNP of swine TLR5.

Development of allele-specific primer PCR for a swine TLR2 SNP and comparison of the frequency among several pig breeds of Japan and the Czech Republic.

In the present study, we have developed an allele-specific primer-polymerase chain reaction (ASP-PCR) for genotyping a single nucleotide polymorphism (SNP) of swine Toll-like receptor 2 (TLR2) (C406G), which is related to the prevalence of pneumonia caused by Mycoplasma hyopneumoniae. We also compared the allele frequency among several pig breeds of Japan and the Czech Republic. Allele-specific primers were constructed by introducing 1-base mismatch sequence before the SNP site. The swine TLR2 C406G mutation was successfully determined by the ASP-PCR using genomic DNA samples in Japan as previously genotyped by a sequencing method. Using the PCR condition determined, genomic DNA samples from pig blood obtained from 110 pigs from 7 different breeds in the Czech Republic were genotyped by the ASP-PCR. The genotyping results from the ASP-PCR were completely matched with the results from the sequencing method. The allele frequency of the swine TLR2 C406G mutation was 27.5% in the Czech Republic and 3.6% in Japan. The C406G mutation was only found in the Landrace breed in Japan, and was almost exclusively found in the Landrace breed in the Czech Republic as well. These results indicated the usefulness of ASP-PCR for detecting a specific SNP for swine TLR2.

Salivary IgA as a useful non-invasive marker for restraint stress in pigs.

In this study, we investigated the expression of immunoglobulin A (IgA) in porcine salivary gland and its relationship with restraint stress in pigs. IgA was expressed in plasma cells in pig salivary gland, as confirmed by immunohistochemical staining. IgA was also detected in pig saliva itself by ELISA, and salivary IgA levels were increased by a restraint stress. Moreover, there was a circadian rhythm of IgA over the course of a day. These results are the first evidence of IgA expression related to stress in the pig saliva and may make IgA useful as a non-invasive biological marker to evaluate acute stress condition in the pigs.

Immune response of gnotobiotic piglets against Mycoplasma hyopneumoniae.

In this study, several cytokine responses were investigated during Mycoplasma hyopneumoniae (Mhp) infection using a gnotobiotic infection model. We found that several inflammatory cytokines (IL-1beta, IL-8, IL-18, and TNF-alpha) and an anti-inflammatory cytokine IL-10 were induced from peripheral blood mononuclear cells (PBMC) of germ-free (GF) piglets stimulated with heat killed Mhp whole antigens, but no IFN-gamma and IL-4 were induced by Mhp. After the intranasal infection of Mhp, IL-1beta, IL-8, IL-18, and IFN-gamma were also detected in the broncho-alveolar lavage fluids (BALF). The antigen-specific IFN-gamma and IL-10 responses after infection of Mhp were gradually suppressed during Mhp infection as well as non-specific immune response to concanavalin A (ConA) and lipopolysacchalide (LPS) at early stage of infection. These results suggested that Mhp infection modulates the immune response of pigs by inducing several cytokines, and causes immuno-suppression of pigs in a gnotobiotic condition.

IL-18 expression in pigs following infection with Mycoplasma hyopneumoniae.

Little is known about the detail of the immune response during infection of pigs with Mycoplasma hyopneumoniae (Mhp). To further understand this important porcine pathogen, we examined the interleukin-18 (IL- 18) response in experimentally infected piglets. We found that large amounts of IL-18 were produced in the bronchoalveolar lavage fluids (BALF) of pigs experimentally infected with Mhp. However, the concentration of interferon-gamma (IFN-gamma) in the same BALF was negatively correlated with that of IL-18. The antibody response against Mhp was found to be associated with the IL-18 concentration in the BALF. Immunohistochemical staining revealed that both IL-18 and IL-18 receptor alpha chain (IL-18Ralpha) were present in macrophages and plasma cells in the lungs of Mhp-infected pigs. Lung mononuclear cells isolated from pneumonic lesions secreted IL-18 and prostaglandin E(2) (PGE(2)) in vitro, and PGE(2) production was enhanced by stimulation with IL-18. These results indicate that IL-18 produced in the pig lung contributes to the development of innate and acquired immune responses against Mhp as a proinflammatory cytokine rather than as an IFN-gamma-inducing factor and may be involved in immunomodulation in pigs.

Bovine IL-18 ELISA: detection of IL-18 in sera of pregnant cow and newborn calf, and in colostrum.

In this study, we examined the concentration of bovine IL-18 in the sera of pregnant cows, their fetuses and newborn calves, and in colostrum in order to examine the role of IL-18 in bovine pregnancy and the neonatal period. A sandwich-ELISA to quantify bovine IL-18 was established using anti-porcine IL-18 monoclonal antibodies, which cross-reacted with bovine IL-18, and used it to measure the concentration of bovine IL-18 in the sera of pregnant cows, their fetuses and newborn calves, and in colostrum. Significant levels of IL-18 were detected in the sera of pregnant cows, but not in the sera obtained from the corresponding fetuses, umbilical arteries and veins. After birth, IL-18 levels in the sera of 1-day and 1-week old calves were low, and significantly increased in the sera of 1-month and 4-month old calves. IL-18 was also detected in colostrum, with the concentration of IL-18 in the first colostrum produced after delivery being the highest, and then decreasing depending on the number of milkings. Furthermore, the serum IL-18 concentration of newborn calves was increased after the oral administration of colostrum. These results suggest that IL-18 during bovine pregnancy and in the newborn period may play important roles in the maintenance of pregnancy and in the maturation of neonatal immunity.

Expression and one-step purification of bovine interleukin-21 (IL-21) in silkworms using a hybrid baculovirus expression system.

A hybrid baculovirus, a hybrid of the Autographa californica nuclear polyhedrosis virus and the Bombyx mori nuclear polyhedrosis virus, was used for the large-scale production of bovine interleukin-21 (IL-21) in silkworms. A recombinant hybrid baculovirus containing the full length of the cDNA of bovine interleukin-21 was constructed and used to infect silkworm larvae or silkmoth pupae. After the infection of the virus, bovine mature IL-21 was produced in the haemolymph or pupal cell lysates. A one-step purification of bovine mature IL-21 from haemolymph using a cation exchange column gave 0.5 mg. IL-21 from 30 ml haemolymph. The bovine IL-21 produced by silkworms strongly induced NK cell proliferation using a human NK cell-line, NK0, and enhanced the lymphokine activated killer (LAK) activity of bovine peripheral blood mononuclear cells.