The genetic organization of the capsular polysaccharide biosynthesis region of Actinobacillus pleuropneumoniae serotype 15

Nucleotide sequence determination and analysis of the cps gene involved in the capsular polysaccharide biosynthesis of Actinobacillus pleuropneumoniae serotype 15 revealed the presence of three open reading frames, designated as cps15ABC genes. At the protein level, Cps15A and Cps15B showed considerably high homology to CpsA (67.0 to 68.7%) and CpsB (31.7 to 36.8%), respectively, of A. pleuropneumoniae serotypes 1, 4 and 12, revealing the common genetic organization of the cps among serotypes 1, 4, 12 and 15. However, Cps15C showed no homology to any proteins of A. pleuropneumoniae serotypes, indicating that cps15C may be specific to serotype 15. This study will provide the basic molecular knowledge necessary for the development of diagnostics and a vaccine for A. pleuropneumoniae serotype 15.     

Porcine CD18 mediates Actinobacillus pleuropneumoniae ApxIII species-specific toxicity

Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, produces Apx toxins that are recognized as major virulence factors. Recently, we showed that ApxIIIA-cytotoxic activity specifically targets Sus scrofa leukocytes. Since both LtxA from Aggregatibacter actinomycetemcomitans (aggressive periodontitis in humans) and LktA from Mannheimia haemolytica (pneumonia in ruminants) share this characteristic, respectively towards human and ruminant leukocytes, and because both use the CD18 subunit to interact with their respective LFA-1, we hypothesized that ApxIIIA was likely to bind porcine CD18 to exercise its deleterious effects on pig leukocytes. A β ₂−integrin-deficient ApxIIIA-resistant human erythroleukemic cell line was transfected either with homologous or heterologous CD11a/CD18 heterodimers using a set of plasmids coding for human (ApxIIIA-resistant), bovine (-resistant) and porcine (-susceptible) CD11a and CD18 subunits. Cell preparations that switched from ApxIIIA-resistance to -susceptibility were then sought to identify the LFA-1 subunit involved. The results showed that the ApxIIIA-resistant recipient cell line was rendered susceptible only if the CD18 partner within the LFA-1 heterodimer was that of the pig. It is concluded that porcine CD18 is necessary to mediate A. pleuropneumoniae ApxIIIA toxin-induced leukolysis.     

Elucidating the role of ApxI in hemolysis and cellular damage by using a novel apxIA mutant of Actinobacillus pleuropneumoniae serotype 10

Exotoxins produced by Actinobacillus (A.) pleuropneumoniae (Apx) play major roles in the pathogenesis of pleuropneumonia in swine. This study investigated the role of ApxI in hemolysis and cellular damage using a novel apxIA mutant, ApxIA336, which was developed from the parental strain A. pleuropneumoniae serotype 10 that produces only ApxI in vitro. The genotype of ApxIA336 was confirmed by PCR, Southern blotting, and gene sequencing. Exotoxin preparation derived from ApxIA336 was analyzed for its bioactivity towards porcine erythrocytes and alveolar macrophages. Analysis results indicated that ApxIA336 contained a kanamycin-resistant cassette inserted immediately after 1005 bp of the apxIA gene. Phenotype analysis of ApxIA336 revealed no difference in the growth rate as compared to the parental strain. Meanwhile, ApxI production was abolished in the bacterial culture supernatant, i.e. exotoxin preparation. The inability of ApxIA336 to produce ApxI corresponded to the loss of hemolytic and cytotoxic bioactivity in exotoxin preparation, as demonstrated by hemolysis, lactate dehydrogenase release, mitochondrial activity, and apoptosis assays. Additionally, the virulence of ApxIA336 appeared to be attenuated by 15-fold in BALB/c mice. Collectively, ApxI, but not other components in the exotoxin preparation of A. pleuropneumoniae serotype 10, was responsible for the hemolytic and cytotoxic effects on porcine erythrocytes and alveolar macrophages.     

The genetic organization of the capsular polysaccharide biosynthesis region of Actinobacillus pleuropneumoniae serotype 14

The genetic organization of the gene involved in the capsular polysaccharide (CPS) biosynthesis of Actinobacillus pleuropneumoniae serotype 14 has been determined. The DNA region for the CPS biosynthesis of serotype 14 (cps14) comprised 9 open reading frames, designated as cps14AB₁B₂B₃CDEFG genes, encoding Cps14A to Cps14G protein, respectively. Cps14A was similar to CpsA of A. pleuropneumoniae serotypes 1, 4 and 12; the Cps14B₁ and Cps14B₂ were similar to CpsB of A. pleuropneumoniae serotypes 1, 4 and 12, suggesting that CPS structure of A. pleuropneumoniae serotype 14 would belong to Group I including A. pleuropneumoniae serotypes 1, 4, 12 and 15. Surprisingly, the overall nucleotide sequence, deduced amino acid sequence, and the genetic organization of the cps14 were nearly identical to those of Actinobacillus suis. This study will provide the molecular basic knowledge for development of diagnostics and vaccine of A. pleuropneumoniae serotype 14.     

Immunological study of an attenuated Salmonella Typhimurium expressing ApxIA,ApxIIA, ApxIIIA and OmpA of Actinobacillus pleuropneumoniae in a mouse model

Salmonella Typhimurium strain expressing the Actinobacillus pleuropneumoniae antigens, ApxIA, ApxIIA, ApxIIIA and OmpA, was previously constructed as a vaccine candidate for porcine pleuropneumonia. This strain was a live attenuated (∆lon∆cpxR∆asd)Salmonella as a delivery host and contained a vector containing asd. An immunological study of lymphocyte proliferation, T-lymphocyte subsets and cytokines in the splenocytes of a mouse model was carried out after stimulation with the candidate Salmonella Typhimurium by intranasal inoculation. The splenic lymphocyte proliferation and the levels of IL-4, IL-6 and IL-12 of the inoculated mice were significantly increased, and the T- and B-cell populations were also elevated. Collectively, the candidate may efficiently induce the Th1- and Th2-type immune responses.     

Enhancement of protective immune responses by oral vaccination with Saccharomyces cerevisiae expressing recombinant Actinobacillus pleuropneumoniae ApxIA or ApxIIA in mice

We previously induced protective immune response by oral immunization with yeast expressing the ApxIIA antigen. The ApxI antigen is also an important factor in the protection against Actinobacillus pleuropneumoniae serotype 5 infection; therefore, the protective immunity in mice following oral immunization with Saccharomyces cerevisiae expressing either ApxIA (group C) or ApxIIA (group D) alone or both (group E) was compared with that in two control groups (group A and B). The immunogenicity of the rApxIA antigen derived from the yeast was confirmed by a high survival rate and an ApxIA-specific IgG antibody response (p < 0.01). The highest systemic (IgG) and local (IgA) humoral immune responses to ApxIA and ApxIIA were detected in group E after the third immunization (p < 0.05). The levels of IL-1β and IL-6 after challenge with an A. pleuropneumoniae field isolate did not change significantly in the vaccinated groups. The level of TNF-α increased in a time-dependent manner in group E but was not significantly different after the challenge. After the challenge, the mice in group E had a significantly lower infectious burden and a higher level of protection than the mice in the other groups (p < 0.05). The survival rate in each group was closely correlated to the immune response and histopathological observations in the lung following the challenge. These results suggested that immunity to the ApxIA antigen is required for optimal protection.     

Pleuritis in slaughter pigs: Relations between lung lesions and bacteriology in 10 herds with high pleuritis

Pleuritis in slaughter pigs has increased in recent years in the Netherlands. The aim of the present study was to determine what respiratory pathogens were involved in pleuritis.In total, lungs of 968 slaughter pigs from 10 herds with high prevalence of pleuritis were morphologically examined for size, location, and type of lesions. Moreover, histology and bacteriology were performed.Examination of gross lung lesions showed 45% pleuritis, 14% pleuropneumonia and 38% catarrhal pneumonia. Peribronchiolar cuffing was found in 61 of 142 samples. Actinobacillus pleuropneumoniae was cultured from 22 lung samples from four herds. Pasteurella multocida was cultured from 55 lung samples in eight herds. No specific pattern with respect to the causal pathogens was found.In conclusion, no single infectious cause of pleuritis was found. A variety of infectious agents combined with environmental factors should be considered as a cause of pleuritis.     

Generation of transgenic corn-derived Actinobacillus pleuropneumoniae ApxIIA fused with the cholera toxin B subunit as a vaccine candidate

Corn, one of the most important forage crops worldwide, has proven to be a useful expression vehicle due to the availability of established transformation procedures for this well-studied plant. The exotoxin Apx, a major virulence factor, is recognized as a common antigen of Actinobacillus (A.) pleuropneumoniae, the causative agent of porcine pleuropneumonia. In this study, a cholera toxin B (CTB)-ApxIIA#5 fusion protein and full-size ApxIIA expressed in corn seed, as a subunit vaccine candidate, were observed to induce Apx-specific immune responses in mice. These results suggest that transgenic corn-derived ApxIIA and CTB-ApxIIA#5 proteins are potential vaccine candidates against A. pleuropneumoniae infection.     

Resistance phenotypes and genotypes among multiple-antimicrobial-resistant Salmonella enterica subspecies enterica serovar Choleraesuis strains isolated between 2008 and 2012 from slaughter pigs in Okinawa Prefecture,Japan

A total of 349 Salmonella enterica subspecies enterica serovar Choleraesuis (S. Choleraesuis) strains, which were isolated between 2008 and 2012 from 349 pigs at two slaughterhouses in Okinawa Prefecture, Japan, were investigated for antimicrobial susceptibility and the presence of antimicrobial resistance genes. All isolates were resistant to at least four antimicrobial agents. The antimicrobial agents for which isolates showed a high incidence of resistance were as follows: ampicillin (100%) and streptomycin (100%), followed by gentamicin (99.7%), oxytetracycline (99.7%), sulfamethoxazole/trimethoprim (99.4%), nalidixic acid (40.1%) and oxolinic acid (40.1%). All isolates were sensitive to cefuroxime, ceftiofur, colistin, fosfomycin, enrofloxacin, orbifloxacin and danofloxacin. The predominant resistance phenotypes and genotypes were: resistance to ampicillin, streptomycin, gentamicin, oxytetracycline and sulfamethoxazole/trimethoprim (58.5%, 204/349) and blaTEM-strA-strB-aadA1-aadA2-aacC2-tet (B)-sul1-sul2-dhfrXII-dhfrXIII (36.1%, 126/349). The quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC and parE of the quinolone-resistant isolates (n=12) showed amino acid substitutions of Ser-83→Phe or Asp-87→Tyr in GyrA and Ser-107→Ala in ParC. To our knowledge, this is the first report on the molecular characterization of antimicrobial resistance among S. Choleraesuis strains in Japan.     

Porcine abortion outbreak associated with Toxoplasma gondii in Jeju Island, Korea

This report deals with the acute onset of an abortion outbreak and high sow mortality in one pig herd consisted of 1,200 pigs and 120 sows on Jeju Island, Korea. Affected pregnant sows showed clinical signs, including high fever, gradual anorexia, vomiting, depression, recumbency, prostration, abortion, and a few deaths. Four dead sows, five aborted fetuses from the same litter, and 17 sera collected from sows infected or normal were submitted to the Pathology Division of the National Veterinary Research and Quarantine Service for diagnostic investigation. Grossly, hepatomegaly and splenomegaly were observed in sows. Multiple necrotic foci were scattered in the lungs, liver, spleen, and lymph nodes. Microscopically, multifocal necrotizing lesions and protozoan tachyzoites were present in the lesions. Tachyzoites of Toxoplasma (T.) gondii were detected immunohistochemically. Latex agglutination showed that the sera of 7 of 17 (41.2%) sows were positive for antibody to T. gondii. The disease outbreak in this herd was diagnosed as epizootic toxoplasmosis. To our knowledge, this is the first report of porcine toxoplasmosis with a high abortion rate and sow mortality in Korea.     

In Vitro Fertilization and Development of Porcine Oocytes Matured in Follicular Fluid

This study was conducted to assess the fertilization and development of porcine oocytes matured in a solo follicular fluid (pFF) using different in vitro culture systems and insemination periods. Cumulus-oocyte complexes (COCs), follicular cells (FCs), and pFF were collected from the follicles of ovaries. The pFF was used as a maturation medium (MpFF) after supplementation with follicle stimulating hormone (FSH) and antibiotics. The COCs were matured in a 15 ml test tube containing 3.5 ml of MpFF with FCs (5.2 × 10⁶ cells/ml; rotating culture system) or 2 ml of MpFF without FCs in a 35-mm petri dish (static culture system) for 44 to 48 h. After maturation culture, oocytes were co-incubated with frozen-thawed spermatozoa for 5 h and then cultured for 7 days. The total mean rates of sperm penetration, normal fertilization, male pronucleus (MPN) formation, cleavage, and development to the blastocyst stage of oocytes after insemination were significantly higher (P<0.01) in the rotating culture system than in the static culture system. In conclusion, compared with the static culture system, the rotating culture system is adequate for the production of developmentally competent porcine oocytes when MpFF is used as a maturation medium.     

Histocytological specificities of adrenal cortex in the New World Monkeys, Aotus lemurinus and Saimiri boliviensis

The New World monkey Aotus spp. (night monkeys) are expected for use of valuable experimental animal with the close species of Saimiri spp. (squirrel monkeys). Saimiri is known to show spontaneous hypercortisolemia, although few reports in Aotus. We compared basic states of blood steroid hormones and histological structure of the adrenal glands in two monkeys. Serum cortisol and ACTH levels were statistically lower in Aotus than Saimiri. Conversely, Aotus adrenocortical area showed significant enlargement, especially at the zona fasciculata. Electron microscopic observation at Aotus fasciculata cells revealed notable accumulation of large lipid droplets and irregular shapes of the mitochondrial cristae. These results suggest potential differences in cellular activities for steroidogenesis between Aotus and Saimiri and experimental usefulness in adrenocortical physiology and pathological models.     

Expression dynamics of bovine MX genes in the endometrium and placenta during early to mid pregnancy

MX belongs to a family of type I interferon (IFN)-stimulated genes, and the MX protein has antiviral activity. MX has at least two isoforms, known as MX1 and MX2, in mammals. Moreover, bovine MX1 has been found to have alternative splice variants—namely, MX1-a and MX1B. In ruminants, IFN-τ—a type I IFN—is temporarily produced from the conceptus before implantation and induces MX expression in the endometrium. However, the expression dynamics of MX after implantation are not clear. In the present study, we investigated the expression of MX1-a, MX1B and MX2 in the endometrium and placenta before and after implantation along with the expression of IFN-α, type I receptors (IFNAR1 and IFNAR2) and interferon regulatory factors (IRF3 and IRF9). Pregnant uterine samples were divided into five groups according to pregnancy days 14–18, 25–40, 50–70, 80–100, and 130–150. Tissue samples were collected from the intercaruncular endometrium (IC), caruncular endometrium (C) and fetal placenta (P). Although all the MX expressions were significantly higher in the IC and C at days 14–18, presumably caused by embryo-secreted IFN-τ stimulation, their expressions were also detectable in the IC, C and P after implantation. Furthermore, IFN-α expression was significantly higher in the IC. RT-PCR indicated IFNAR1, IFNAR2, IRF3 and IRF9 mRNA in all the tissues during pregnancy. These results suggest that all the MX genes are affected by the type I IFN pathway during pregnancy and are involved in an immune response to protect the mother and fetus.     

Laser induced injury caused hyperglycemia-like effect in Drosophila larva: a possible insect model for posttraumatic diabetes

Diabetic patients need particular care in case of infection, digestive disorder or external injury, because external stress often exasperates the glucose metabolism, which is known as “sick day management”. In addition, severe trauma can be a cause of hyperglycemia with insulin resistance. In spite of critical component of the treatment, the precise mechanisms of how trauma develops posttraumatic diabetes remain unknown. Here, we ablated body wall muscles in Drosophila larvae by laser beam and found that the level of trehalose, the principal sugar circulating in the hemolymph or in the tissues of most insects, was increased. The model may provide a helpful tool to understand the relationship between trauma and sugar metabolism.     

Disease patterns and immune responses in the offspring to sows with high or low antibody levels to Actinobacillus pleuropneumoniae serotype 2

The serum antibody responses to Actinobacillus pleuropneumoniae and the secondary invader Pasteurella multocida were monitored from birth until slaughter in the offspring to sows with high or low levels of serum antibodies to A. pleuropneumoniae.Serum antibody concentrations to A. pleuropneumoniae were higher from birth to the age of 9 weeks in piglets delivered by high responding sows. In contrast, antibody levels to P. multocida were similar in both groups during this period. From the age of 20 and 15 weeks, antibody levels to A. pleuropneumoniae and P. multocida, respectively, were higher in the offspring to high responding sows.This implies that the offspring to sows with high levels of antibodies may be better protected during the first period of life because of a higher level of passively derived immunity. These piglets will also mount a higher antibody response when later infected, indicating a heritability of the humoral immune response.