Evaluation of an indirect enzyme-linked immunosorbent assay (ELISA) using recombinant toxin for detection of antibodies against Pasteurella multocida toxin

To facilitate the control of progressive atrophic rhinitis (PAR) of swine caused by toxigenic Pasteurella multocida, an enzyme-linked immunosorbent assay (ELISA) and a serum neutralization test (NT) have recently been developed to detect antibodies against the P. multocida dermonecrotic toxin (PmDNT). However, the NT is a cumbersome and time-consuming technique. To overcome these drawbacks, we developed an indirect ELISA, using recombinant PmDNT expressed in Escherichia coli, for the detection of antibodies to PmDNT in serum samples from pigs. The practical usefulness of this ELISA was compared with the NT using serum samples obtained from experimentally infected and naturally infected pigs. In the pigs experimentally inoculated with vaccine including PmDNT toxoid, the ELISA and neutralization antibodies were detected at almost the same time, and a good correlation was demonstrated between both tests (P<0.01, R(2)=0.807). Therefore, the ELISA can be used to evaluate the immune reaction of pigs after vaccination with P. multocida toxoid. In a survey conducted on a field herd with a history of clinical AR, the seropositivity by ELISA in pigs of age 4.5-6 months was increased even though the NT was negative, and the correlation was low between the results obtained with the two tests (P<0.01, R(2)=0.38). Therefore, the results indicated that this ELISA might be a useful alternative to the NT currently used to detect the antibody to PmDNT after vaccination or infection with P. multocida.     

Detection of Mycoplasma hyopneumoniae in lung and nasal swab samples from pigs by nested PCR and culture methods

We examined nasal swab and lung homogenate samples collected from pigs experimentally and naturally infected with Mycoplasma hyopneumoniae for the detection of M. hyopneumoniae by the nested PCR (nPCR) and culture methods. In the 23 experimentally infected pigs, M. hyopneumoniae was commonly detected in nasal swabs by the nPCR and culture methods at 4 weeks after inoculation, and there was a significant correlation (P<0.01) between the titers of viable organisms in nasal swabs and in lung homogenates in the experimentally inoculated pigs. In the naturally infected pigs, on the other hand, discrepancies in detection were found between nasal swab and lung homogenate samples in 17 of 36 cases, although the presence of gross lung lesions correlated relatively well with the detection of organisms from the samples. Our results indicated that the diagnosis of mycoplasmal pneumonia by nPCR in individual pigs with nasal swabs is reliable under these experimental conditions. At present, nPCR with nasal swabs should only be used for monitoring the disease status at the herd level under field conditions.     

Transmaxillary anchored silicon embedded gauze plug in the post-operative treatment of a large oromaxillary fistula caused by a supernumerary cheek tooth

A 9-year-old Hungarian sport horse gelding was presented to the clinic in poor condition displaying malodorous bilateral purulent nasal discharge. Oral examination revealed the presence of supernumerary 111 and 211. Bilateral diastema formation between the third maxillary molars and the supernumerary teeth with deep periodontal pockets and massive food impactions were diagnosed endoscopically. Radiography revealed inhomogenous sinus opacities in the left and right paranasal sinuses. Following bilateral oral extractions of the supernumerary cheek teeth and third maxillary molars, bilateral oromaxillary fistula formations were diagnosed (about 17 mm diameter on the right side). Bilateral frontal and right-sided maxillary trephinations and resection of the right bulla of the maxillary septum were performed. Massive food impactions of the left and right paranasal sinuses were removed under endoscopic control. Repeated trans-endoscopic sinus lavage was performed post-operatively. After 2.5 months, the large right-sided oromaxillary communication was temporarily closed with a transmaxillary anchored, gauze-cored silicon plug on an outpatient basis. Follow-up examination after 187 days revealed complete closure of the oromaxillary fistula and absence of sinusitis. In a 1.5-year follow-up control, no pathological conditions were found.     

Serological diagnosis of enzootic pneumonia of swine by a double-sandwich enzyme-linked immunosorbent assay using a monoclonal antibody and recombinant antigen (P46) of Mycoplasma hyopneumoniae

To facilitate the control of enzootic pneumonia (EP) of swine caused by Mycoplasma hyopneumoniae, the complement fixation (CF) test has been used for the detection of M. hyopneumoniae antibodies. However, the CF test is a cumbersome and time-consuming technique and cross-reactivity are major drawbacks associated with this method. To circumvent these drawbacks, we have developed a double-sandwich enzyme-linked immunosorbent assay (ELISA), consisting of purified monoclonal antibody (Mab) against the 46 kDa surface antigen (P46) of M. hyopneumoniae and recombinant P46 protein expressed in Escherichia coli, for the detection of antibodies to M. hyopneumoniae in serum samples from pigs experimentally inoculated with M. hyopneumoniae and from naturally infected pigs, and compared the practical usefulness of ELISA using the CF test. In experimentally inoculated pigs, the CF and ELISA antibodies were detected at almost the same time, and a good correlation was demonstrated between the CF test and the ELISA. In a survey conducted on field samples, the seropositivity by ELISA in pigs of age 2-6 months was increased. At the time of slaughter, approximately 80% of the animals were seropositive for ELISA. However, a gradual decrease in the prevalence of ELISA positive samples was observed in sows with increasing parity. No correlation was seen between the results obtained with the two methods in the clinical samples. The CF test appears to have limited value for the diagnosis of EP in conventional herds because nonspecific reactions were frequently observed. Therefore, this ELISA is a useful alternative to the CF test currently used for the diagnosis of EP.     

Automatic counting of FISH-labeled microbes by an LED illuminated detecting apparatus

ABSTRACT:   The Bioplorer (BP) (Matsushita Ecology Systems Co. Ltd, Kasugai, Aichi-ken, Japan) is an apparatus that consists of a light-emitting diode and optical and image analysis systems. This instrument has been developed and used for the semi-automatic counting of total microbes, mainly in food, cosmetics and industrial products. In this study, the applicability of BP to the detection and enumeration of eukaryotic and prokaryotic cells, after the fluorescent in situ hybridization (FISH) procedure, was examined. The cells of a yeast ( Candida albicans ) and a bacterium ( Escherichia coli ) were specifically labeled using an oligonucleotide probe with fluorochrome and then counterstained with 4', 6-diamidino-2-phenylindole (DAPI). The numbers of cells after FISH treatment and DAPI staining agreed well, indicating that the BP optical device and image analysis system had enough sensitivity to detect and quantify eukaryotic and prokaryotic cells. Because of its simplicity and reliability, BP can be a new tool for quantification of specifically labeled target microorganisms, both in industrial products and natural samples.     

Evaluation of a new eastern blotting technique for the analysis of ginsenoside Re in American ginseng berry pulp extracts

A new eastern blotting technique has been established for ginsenoside Re (G-Re) contained in American ginseng berry pulp extracts. G-Re in American ginseng berry pulp was extracted using 100% methanol, 100% ethanol, 50% aqueous methanol, and 50% aqueous ethanol. The combined crude extracts were applied onto a polyethersulfone membrane and developed using the methanol–water–acetic acid solvent system (45:55:1 v/v). Separated components were immunostained using anti-G-Re monoclonal antibody. G-Re was first specifically detected and then quantitatively analyzed using NIH Imaging software. We also confirmed that the most suitable solvent was 50% aqueous methanol for extracting G-Re from American ginseng berry pulp.     

Enzyme-linked immunosorbent assay for total sennosides using anti-sennside A and anti-sennoside B monoclonal antibodies

Total sennosides concentration is a very important factor when rhubarb and senna will be used as crude drugs. However, one-step analytical technique for total sennosides has not been reported except HPLC. An enzyme-linked immunosorbent assay (ELISA) for total sennosides concentration by using the combination of anti-sennoside A (SA) and anti-sennoside B (SB) monoclonal antibodies (MAbs) in a single assay has been investigated. Total sennosides concentration in rhubarb and senna samples determined by newly developed assay system showed good agreement with those analyzed by ELISA using anti-SA MAb and anti-SB MAb, respectively.