Development of Actinobacillus pleuropneumoniae ApxI, ApxII, and ApxIII-specific ELISA methods for evaluation of vaccine efficiency

Among various vaccines against Actinobacillus pleuropneumoniae, subunit vaccines using recombinant proteins of ApxI, ApxII, and ApxIII as vaccine antigens have shown good efficacy in terms of safety and protection. Therefore, subunit vaccines are being applied worldwide and the development of new subunit vaccines is actively being conducted. To evaluate the efficacy of the subunit vaccines, it is important to measure immune responses to each Apx toxin separately. However, the cross-reactivity of antibodies makes it difficult to measure specific immune reactivity to each toxin. In the present study, specific antigen regions among the toxins were identified and cloned to solve this problem. The antigenicity of each recombinant protein was demonstrated by Western blot. Using the recombinant proteins, we developed enzyme-linked immunosorbent assay (ELISA) methods that can detect specific immune responses to each Apx toxin in laboratory guinea pigs. We suggest that the ELISA method developed in this study can be an important tool in the evaluation of vaccine efficiency and vaccine development.

Characterization of Specific IgA Response to Antigenic Determinants of Helicobacter pylori Urease Encoded by ureA and ureB in Children

Helicobacter pylori (H. pylori), a causative agent of chronic gastritis and gastric cancer, has several virulent factors for own survival and progression toward gastric diseases in human stomach. Of those, H. pylori produces mainly urease (10~15% total protein weight) that neutralize the gastric acid for survival. Here, we identified the antigenic epitope of urease and then developed an ELISA using the antigen including the epitope of urease. We identified the antigenic epitope of urease that induces IgA antibodies in human using truncated mutants. Eight kinds of serially-truncated mutant of UreA and UreB were prepared and subjected to immunoblot using pooled sera of patients with gastric disorders. UreBEnd protein containing UreB epitope was produced and investigated its diagnostic value via ELISA in children. As a result, mutants having last 24 amino acid residues of UreB carboxyl terminus deleted did not show IgA-reactive band. The clones that contained the downstream of 448(th) amino acid in UreB showed IgA-reactive band. The serodiagnostic value of the UreBEnd recombinant protein including identified epitope was confirmed via IgA ELISA and shown to have 97% sensitivity and 100% specificity. These results demonstrated that carboxyl terminal region of UreB carries an antigenic epitope for IgA response in human. It may be useful for detecting H. pylori infection with improved test accuracy and minimum use of endoscopy.

Gene Expression Profiles of Th1-type Chemokines in Whole Blood of Mycobacterium avium subsp. paratuberculosis-Infected Cattle

Johne's disease (JD) is a chronic, debilitating disease of ruminants including cows, and is caused by Mycobacterium avium subsp. paratuberculosis (MAP). MAP is not only important in animal husbandry, but also in public health as it is associated with the onset of Crohn's disease, a chronic inflammatory bowel disease in humans. JD, like other mycobacterial diseases including tuberculosis, is classified into different stages based on the progression of infection. In addition, development of diagnostic assays that can distinguish between subclinical and clinical stages of JD is essential to control mycobacterial infection by providing an effective treatment. For the development of novel diagnostic methods of JD, it is important to investigate and understand the mRNA expression of the various immune markers in individuals at each stage of infection. In this study, we measured the levels of Th1-type chemokines, CXCR3, CCL4, CCL5, CXCL9, CXCL10, and CXCL11 in MAP-infected bovine blood by interferon (IFN)-γ release assay (IGRA) using IFN-γ as an alternative biomarker. The association of mRNA expression patterns of these chemokines with the MAP infection stages was analyzed and IFN-γ, CCL5, and CXCL10 were found to be significantly upregulated compared to IFN-γ, the biomarker used in IGRA. Our results further indicate that IFN-γ levels significantly increased in individuals with MAP-specific antibody, and CCL5 and CXCL10 levels significantly increased in those with MAP DNA. In particular, CCL5 was significantly upregulated in individuals, in which both MAP-specific antibody and MAP DNA were detected, but the expression of CXCL10 was specifically elevated in MAP DNA-detected individuals without MAP-specific antibody.

ArmA and RmtB Were the Predominant 16S RMTase Genes Responsible for Aminoglycoside-resistant Isolates in Korea

Pathogenic gram-negatives that produce 16S ribosomal RNA methyltransferases (16S RMTases) have already been distributed all over the world. To investigate the predominance of aminoglycoside resistance associated with 16S RMTases in Korea, we collected a total of 222 amikacin resistant Gram-negative clinical isolates from patient specimens between 1999 and 2015 from three hospital banks across Korea. ArmA and rmtB were the predominant 16S RMTase genes responsible for aminoglycoside-resistant isolates circulating in Korean community settings although only one rmtA-producing isolate was detected in 2006.

Isolation and Genetic Characterization of Orientia tsutsugamushi from Scrub Typhus Patients in Gyeongsangnam-do, Korea

Orientia tsutsugamushi (O. tsutsugamushi), which is endemic to an Asia-Pacific region, has increased its incidence and caused annually around 10 thousand patients infected with scrub typhus in Korea in the past several years. In the present study, we isolated 44 O. tsutsugamushi from the patients with febrile illness accompanied with or without an eschar in Gyeongsangnam-do, Korea. These isolates were characterized by genetic analysis of the major outer membrane protein, the 56-kDa type-specific antigen (tsa56), which is unique to O. tsutsugamushi. Two types of sequences of tsa56, designated by JJ1 and JJ2, were determined from 37 and 7 isolates of the 44 isolates, respectively. JJ1 and JJ2 showed 74.7~90.8% identity in nucleotide sequence and 66.1~90.5% identity in amino acid sequence with 33 reference strains except for Boryong and Kuroki. JJ1 and JJ2 had 100 and 99.9% nucleotide identity to Boryong strain, and 99.9 and 99.8% to Kuroki, which has been known to be similar to Boryong, respectively. In addition, they showed 77.9~ 81.4% nucleotide identity with the cluster of Gilliam-related genotypes, whereas they showed higher nucleotide identity (89.6~90.8%) with the cluster of Karp-related genotypes. To our knowledge, this is the first report to isolate O. tsutsugamushi and characterize their genotype as the Boryong in Jinju and West Gyeongsangnam-do, Korea, even though it has been reported that the Boryong was the predominant genotype in isolates from chiggers, domestic rodents, and patients in the southern part of Korea. Furthermore, our isolates could be useful source to study on the pathophysiology and epidemiology of scrub typhus in Korea.

Discovery of antigens for early detection of Mycobacterium avium subsp. paratuberculosis and analysis of characteristics using bioinformatics tools / 대한수의학회지

Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is one of the most widespread and economically important diseases in cattle. Current diagnostic methods are based on the detection of anti-MAP antibodies in serum or isolation of the causative agent. However, these techniques are often not applicable for cases of subclinical infection due to relatively low sensitivity. Therefore, finding new antigen candidates that strongly react with the host immune system had been attempted. To effectively detect infection during the subclinical stage, several antigen candidates were selected based on previous researches. Characteristics of the selected antigen candidates were analyzed using bioinformatics-based prediction tools. A total of nine antigens were selected (MAP0862, MAP3817c, MAP2077c, MAP0860c, MAP3954, MAP3155c, MAP1204, MAP1087, and MAP2963c) that have MAP-specific and/or high immune responses to infected animals. Using a transmembrane prediction tool, five of the nine antigen candidates were predicted to be membrane protein (MAP3817c, MAP3954, MAP3155c, MAP1087, and MAP1204). Some of the predicted protein structures identified using the I-TASSER server shared similarities with known proteins found in the Protein Data Bank database (MAP0862, MAP1204, and MAP2077c). In future studies, the characteristics and diagnostic efficiency of the selected antigen candidates will be evaluated.

Virulence factors, antimicrobial resistance patterns, and genetic characteristics of hydrogen sulfide-producing Escherichia coli isolated from swine / 대한수의학회지

Escherichia (E.) coli is commensal bacteria found in the intestine; however, some pathogenic strains cause diseases in animals and humans. Although E. coli does not typically produce hydrogen sulfide (H2S), H2S-producing strains of E. coli have been identified worldwide. The relationship between virulence and H2S production has not yet been determined. Therefore, characteristics of H2S-producing isolates obtained from swine feces were evaluated including antibiotic resistance patterns, virulence gene expression, and genetic relatedness. Rates of antibiotic resistance of the H2Sproducing E. coli varied according to antibiotic. Only the EAST1 gene was detected as a virulence gene in five H2S-producing E. coli strains. Genes conferring H2S production were not transmissible although the seeA gene encoding 3-mercaptopyruvate sulfurtransferase was detected in all H2S-producing E. coli strains. Sequences of the seeA gene motif CGSVTA around Cys238 were also identical in all H2S-producing E. coli strains. Diverse genetic relatedness among the isolates was observed by pulsed-field gel electrophoresis analysis. These results suggested that H2S-producing E. coli strains were not derived from a specific clone and H2S production in E. coli is not associated with virulence genes.

Assessing PCV2 antibodies in field pigs vaccinated with different porcine circovirus 2 vaccines using two commercial ELISA systems

Porcine circovirus type 2 (PCV2) is the primary causative agent for post-weaning, multisystemic, wasting syndrome. Consequently, serologic detection of and vaccination against PCV2 are important for the swine industry. Among several serological tests, the enzyme-linked immunosorbent assay (ELISA) is commonly used to measure anti-PCV2 antibody levels. In the present study, we used two commercial ELISA systems to comparatively evaluate anti-PCV2 antibodies in field pigs treated with three different PCV2 vaccines. Among a total of 517 serum samples, the results of the two ELISAs were fully concordant for 365 positive and 42 negative samples, indicating 78.7% agreement. In addition, the Pearson coefficient (0.636) indicated a moderate correlation between data from the two ELISAs. Results from the farms with pigs vaccinated with the three different PCV2 vaccines demonstrated that most of the vaccinated animals underwent seroconversion. However, the increase and duration of antibody titers varied depending on the vaccine, the presence of maternal antibodies, and the vaccination program. PCV2 serologic status and anti-PCV2 antibody levels of herds from this study could be utilized to determine the best timing for vaccination and assessing vaccination compliance.

Effective DNA extraction method to improve detection of Mycobacterium avium subsp. paratuberculosis in bovine feces / 대한수의학회지

Paratuberculosis caused by Mycobacterium avium subsp. paratuberculosis (MAP) has extended latent periods of infection. Due to this property, difficulties in the detection of fecal shedder have been raised. A newly designed method for DNA extraction from fecal specimens, mGITC/SC was evaluated in terms of diagnostic efficiency. The detection limit of IS900 real-time PCR was about 50 MAP (1.5 cfu) in 250 mg of feces (6 cfu per g). Also, this DNA extraction method was faster and cheaper than that using commercial kit or other methods. Consequently, the mGITC/SC is an economical DNA extraction method that could be a useful tool for detecting MAP from fecal specimens.

Development of a novel enzyme-linked immunosorbent assay to detect anti-IgG against swine hepatitis E virus

Swine hepatitis E virus (HEV) is widespread throughout pigs in both developing and industrialized countries. This virus is an important zoonotic agent and a public concern worldwide. Infected pigs are asymptomatic, so diagnosing swine HEV relies on detection of the virus or antibodies against the virus. However, several obstacles need to be overcome for effective and practical serological diagnosis. In this study, we developed an enzyme-linked immunosorbent assay (ELISA) that used a purified recombinant capsid protein of swine HEV. The potential clinical use of this assay was evaluated by comparing it with a commercial kit (Genelabs Technologies, Diagnostics, Singapore). Results of the ELISA were highly correlated with those of the commercial kit with a sensitivity of 97% and specificity of 95%. ROC (receiving operator characteristic) analysis of the ELISA data produced a value of 0.987 (95% CI, 0.977~0.998, p < 0.01). The cut-off value for the ELISA was also determined using negative pig sera. In summary, the HEV-specific ELISA developed in the present study appears to be both practical and economical.

Application of SYBR Green real-time PCR assay for the specific detection of Salmonella spp / 대한수의학회지

The aim of this study was to applicate and evaluate a SYBR Green real-time PCR for the specific detection of Salmonella spp. Specificity of the PCR method was confirmed with 48 Salmonella spp. and 5 non-Salmonella strains using invA gene primer. The average threshold cycle (C(T)) of Salmonella spp. was 11.83 +/- 0.78 while non-Salmonella spp. was 30.86 +/- 1.19. Correlation coefficients of standard curves constructed using C(T) versus copy number of Salmonella Enteritidis ATCC 13076 showed good linearity (R2 = 0.993; slope = 3.563). Minimum level of detection with the method was > 10(2) colony forming units (CFU)/mL. These results suggested that the SYBR Green real-time PCR might be applicable for the specific detection of Salmonella spp. isolates.

Field evaluation of Enterotoxigenic Escherichia coli-specific bacteriophage (PhiCJ19) as a feed additive / 대한수의학회지

Field efficacy of enterotoxigenic Escherichia coli-specific phage (PhiCJ19) as a feed additive was evaluated in weaning piglets. Fifty-four piglets at 3~4 weeks old were allocated in three different groups and two of them were fed with bacteriophage at different concentrations (10(6) PFU/kg feed and 10(8) PFU/kg feed, respectively) for 30 days. Body weight and feed intake were measured at 10 days interval and body condition and fecal score were inspected every day. Based on the measurement, feed conversion rate (FCR) and average daily gain (ADG) of each group during 30 days were analyzed. The analysis suggests that the bacteriophage may help the improvement of FCR and ADG at 10(8) PFU/kg of bacteriophage feeding group in 30 days. A result from analysis of fecal score indicates that the bacteriophage also may help to relieve the intermittent diarrhea in post-weaning stage. Those results suggest that bacteriophage might help the growth of piglets in post-weaning stage.

The effects of Germanium biotite on the adsorptive and inhibition of growth abilities against E. coli and Salmonella spp. in vitro / 대한수의학회지

Germanium biotite, a natural mineral, has been used as a feed supplement to reinforce innate immune ability. The aim of the present study was to evaluate the effects of germanium biotite on the adsorptive and inhibition of growth abilities against Escherichia (E.) coli and Salmonella spp. in vitro. Two strains of enterotoxigenic E. coli and four strains of two Salmonella serotypes (Salmonella Derby and Salmonella Typhimurium), major bacterial diarrheal pathogens, were used for this experiment. The absorptive ability of germanium biotite against most Salmonella used in present experiment was observed weakly. The germanium biotite, however, showed significant effect of bacterial growth inhibition in most experiment bacteria. These results suggest that the use of the germanium biotite as feed supplement could alleviate diarrhea following inhibition of bacteria growth. It is also presumed that antibiotics usage for farm animals, considered as causes of antibiotic residue in meat and emerging antibiotic resistance, could be reduced through the use of germanium biotite as a feed supplement, in place of antibiotics used for the prevention of diarrhea.

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.

Comparison of tuberculin skin test with Interferon-gamma assay for the diagnosis of bovine tuberculosis in Korean cattle / 대한수의학회지

Bovine tuberculosis (bTB), caused primarily by Mycobacterium bovis, continues to exert an economic loss, even in countries with active control measures, and is one of zoonotic diseases enable to be transmitted to human. The control and eradication of bTB are mainly based on a test and slaughter policy and/or abattoir surveillance. Various factors including limitation of diagnostic tests have been considered as major constraints to eradication. Single intradermal test (SIT) is the official diagnostic test. New diagnostic methods are needed to be developed, because of limitations of the test. In the present study SIT was compared with single intradermal comparative cervical test (SICCT) and interferon (IFN)-gamma assay. There was very low correlation between SIT and SICCT. However, high correlation was shown between SIT and IFN-gamma assay while no correlation was observed between SICCT and IFN-gamma assay. Therefore, our results suggest the possibility of replacement of SIT with IFN-gamma assay for the diagnosis of bovine tuberculosis.

The expression and cellular localization of phospholipase D isozymes in the developing mouse testis

To examine the involvement of phospholipase D (PLD)isozymes in postnatal testis development, the expression ofPLD1 and PLD2 was examined in the mouse testis atpostnatal weeks 1, 2, 4, and 8 using Western blot analysisand immunohistochemistry. The expression of both PLD1and PLD2 increased gradually with development frompostnatal week 1 to 8. Immunohistochemically, PLDimmunoreactivity was detected in some germ cells in thetestis and interstitial Leydig cells at postnatal week 1.PLD was mainly detected in the spermatocytes andresidual bodies of spermatids in the testis after 8 weeksafter birth. The intense immunostaining of PLD in Leydigcells remained unchanged by postnatal week 8. Thesefindings suggest that PLD isozymes are involved in thespermatogenesis of the mouse testis.