Risk factors for bovine anthrax in Bangladesh, 2010-2014: a case-control study.

A matched case-control study was conducted in Bangladesh by enrolling case smallholdings of cattle affected with anthrax in the period of October 2010 to December 2014. The cases were initially reported by mass media and/or in surveillance reports from authorities concerned in the country. In total, 43 case smallholdings were enrolled. For each case, a control was matched by similarity in herd-size and rearing of animals, selected from a distantly located (within 3-10 km) place but within the same sub-district of the case farm. Data collected by administering a prototype questionnaire were analysed by matched-pair analysis and multivariable conditional logistic regression. Out of the 43 smallholdings, 41 were located in three adjoining districts: Pabna, Sirajganj and Tangail, apparently forming a spatial cluster, could be termed 'anthrax hot spot' in Bangladesh. Sick animal on farm or a nearby farm slaughtered in the recent past (odds ratio (OR) 12.2, 95% confidence interval (CI) 1.6-93.4, P = 0.016)), history of heavy rains occurring in the last 2 weeks preceding an outbreak (OR 13.1, 95% CI 1.2-147.1, P = 0.037) and disposing of dead animal into nearby water body (OR 11.9, 95% CI 1.0-145.3, P = 0.052) were independent risk factors for anthrax in cattle in the country.

Prevalence of Metallo-ß-lactamase Producing Non-fermentative Pseudomonas Species from Clinical Isolates in Dhaka, Bangladesh.

Antimicrobial drug resistance, a global concern, has been increasing unpredictably in microorganism causing human infections specially among Gram negative non-fermenting Pseudomonas spp. Carbapenems, a beta lactam antibiotics, are the most potent and effective drug usually kept reserved for treating the multi-drug resistant Psedomonas spp and other infections caused by organisms producing Extended Spectrum Beta Lactamase (ESBL) and AmpC. Clinical utility of carbapenem will reduce when resistant bacteria evolve due to production of carbapenem hydrolyzing Metallo-ß-lactamase (MBL) which confers high-level resistance to all beta-lactam antibiotics except aztreonam. The various reports on the prevalence of MBLs are available from many countries but few from Bangladesh. We investigated the prevalence of MBL production in these Pseudomonads obtained from clinical sources in an uraban setting in Dhaka, Bangladesh. A total of 29,136 specimens were processed for culture from January 2011 and December 2015 from non duplicated patients attending diagnostic unit of icddr,b from different settings of Bangladesh. The specimens included urine 14,323; blood 11,378; other body fluid 2,487; sputum 535 and tracheal aspirate 413. All specimens were processed for culture following standard bacteriological methods and the Pseudomonas spp were identified following defined standard biochemical procedures. Metallo-ß-lactamase (MBL) was determined by EDTA disk synergy (EDS) test. Antimicrobial susceptibility test was performed by disk diffusion method and susceptibility pattern was interpreted and reported following Clinical Laboratory Standard Institute (CLSI) guideline. From 29,136 specimens a total of 2,340(8%) were isolated and identified as Pseudomonas spp. Of the identified Pseudomonas spp, 238(57.6%) were from tracheal aspirate, 216(40.4%) from sputum, 902(36.7%) from other body fluids, 463(4.1%) from blood and 521(3.6%) from urine samples. From 2,340 Pseudomonas spp, by selective sampling, imipenem-meropenem resistant and intermediate susceptible 100 strains were tested for MBL production and 92 were found positive. Tracheal aspirate showed 38%, other body fluids 30%, Urine 17%, sputum 4% and blood 3% MBL production respectively. Irrespective of the sources of specimens, Pseudomonas spp showed 71% resistance to cefixime, 70% to ceftriaxone, 64% to gentamicin, 56% to piperecillin+tazobactam, 50% to ciprofloxacin, 49% to amikacin, 46% to netilmicin, 45% to ceftazidime, 30% to meropenem, 26% to imipenem and 19% to polymyxin B. As multi-drug resistant Pseudomonas showed high level of (92%) MBL production, so MBL detection testing facility may be a useful battery to determine MDR producing Pseudomonas from clinical isolates.

Influence of Detection Methods in Characterizing Escherichia coli O157:H7 in Raw Goat Meat Using Conventional and Molecular Methods.

　Presence of Escherichia coli O157:H7 on fresh goat meat samples (n= 40) of Dhaka city was analyzed using conventional and molecular methods. A total of 86 presumptive E. coli O157:H7 colonies were isolated from 60% of the samples using selective agar plating method. After conventional biochemical assay followed by API 20E assay, only 11 isolates were found to be E. coli O157:H7. Further serological test identified only four isolates that has strong agglutination reaction against anti-H7 sensitized latex. The biochemically and serologically confirmed isolates were then screened for major virulence factors include eaeA, rfbE, fliC, stx1 and stx2 genes by PCR. PCR analysis of positive isolates showed, 10 isolates were eaeA and rfbE genes positive but fliC gene was only in six, indicating that these isolates were H7 positive with flagellum antigens which might not expressed or detected in serotyping tests. Multiplex PCR against eaeA, stx1 and stx2 genes of the isolates showed similar results as when done individually. These results revealed that only 7% of the primary presumptive E. coli O157:H7 was found to be stx producing E. coli O157:H7 and thus greatly influenced the detection of the pathogen in meat samples.

Evaluation of efficiency of nested multiplex allele-specific PCR assay for detection of multidrug resistant tuberculosis directly from sputum samples.

UNLABELLED: For an effective control of tuberculosis, rapid detection of multidrug resistant tuberculosis (MDR-TB) is necessary. Therefore, we developed a modified nested multiplex allele-specific polymerase chain reaction (MAS-PCR) method that enables rapid MDR-TB detection directly from sputum samples. The efficacy of this method was evaluated using 79 sputum samples collected from suspected tuberculosis patients. The performance of nested MAS-PCR method was compared with other MDR-TB detection methods like drug susceptibility testing (DST) and DNA sequencing. As rifampicin (RIF) resistance conforms to MDR-TB in greater than 90% cases, only the presence of RIF-associated mutations in rpoB gene was determined by DNA sequencing and nested MAS-PCR to detect MDR-TB. The concordance between nested MAS-PCR and DNA sequencing results was found to be 96·3%. When compared with DST, the sensitivity and specificity of nested MAS-PCR for RIF-resistance detection were determined to be 92·9 and 100% respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: For developing- and high-TB burden countries, molecular-based tests have been recommended by the World Health Organization for rapid detection of MDR-TB. The results of this study indicate that, nested MAS-PCR assay might be a practical and relatively cost effective molecular method for rapid detection of MDR-TB from suspected sputum samples in developing countries with resource poor settings.

The Mycobacterium bovis BCG 64-kDa surface protein is antigenically shared with different mouse tumor cells and has anti-tumor activity in immunized mice.

The Mycobacterium bovis BCG 64-kDa surface protein, which was found to share antigenic determinants with line 10 hepatoma cells and showed anti-line 10 tumor activity in immunized guinea pigs, has also been found to share common antigenic determinants with Meth A, CT-26 and RL female 1 mouse tumor cells. The 64-kDa protein also demonstrated anti-tumor activity in immunized mice and 37% of the animals challenged with Meth A tumor cells and 50% of those challenged with CT-26 tumor cells completely rejected further tumor growth in the immunized mice. All these data clearly suggest that BCG 64 kDa protein is probably identical with the tumor specific antigen.

Crossed immunoelectrophoretic analysis of antigenic composition of B-subunit/whole-cell and whole-cell only killed oral cholera vaccines.

Crossed immunoelectrophoresis was used to identify antigens preserved in the whole-cell component of oral cholera vaccines tested in the field trial in Bangladesh. The composition and immunogenicity of the vaccine antigens were compared with those of antigens obtained from live cells of Vibrio cholerae 01 of both biovars and serovars. The whole-cell component of the vaccine contained ten antigens in comparison with the live Vibrio cells which revealed the presence of 30 antigens. The whole-cell component contained lipopolysaccharide, flagellar antigen, one cell-bound haemagglutinin and at least six outer membrane protein antigens.

Enterotoxicity ofVibrio furnissii isolated from eels.

Fourteen strains ofVibrio furnissii, isolated from different ulcerated areas of eel, were tested to check their enterotoxicity in an animal model. Most strains caused fluid accumulation in ileal loop tests after serial passages and culture filtrates of most of the strains caused induration and increase in vascular permeability in rabbit skin. Production of extracellular haemolysin was also detected in all the culture filtrates. All of these observations clearly establish the enterotoxicity of these organisms.

A 64 kDa protein from Mycobacterium bovis BCG shares the same antigenic determinants with line 10 hepatoma cells and has anti-line 10 tumor activity.

A 64 kilodalton (kDa) surface protein was isolated from the water-extracted materials from Mycobacterium bovis strain BCG, the determinants of which are antigenically shared by a 64 kDa major surface antigenic component of line 10 hepatoma cells. The 64 kDa protein showed anti-line 10 tumor activity in pre-immunized guinea pigs, and this suggest that the BCG 64 kDa protein is probably identical with the tumor specific antigen.

Idiotype vaccine for tumor by anti-idiotypic antibody prepared against anti-(bacillus Calmette Guèrin)BCG monoclonal antibody.

The anti-idiotypic antibody (Ab2) prepared against the anti-BCG monoclonal antibody (mAb) (Ab1) exhibited potential vaccine activity against Meth A fibrosarcoma that shared a common antigen(s) with Mycobacterium bovis strain bacillus Calmette Guèrin (BCG). Mice vaccinated with the anti-idiotypic antibody (Ab2) were protected significantly against growth of the transplanted Meth A tumor (66%), and the presence of anti-(anti-idiotypic antibody) (Ab3) was proved in the Ab2-vaccinated mice by enzyme-linked immunosorbent assay and indirect immunofluorescence analyses using unabsorbed or absorbed sera against the BCG antigen(s) and Meth A tumor cells. This indicated that the anti-idiotypic antibody (Ab2) mimicked the structures of the BCG antigen(s) and behaved as the BCG antigen(s) to induce the Ab1-like antibody (Ab3) in vivo. Presumably the Ab2-induced Ab3 plays a significant role in preventing growth of the transplanted tumor in animals. By contrast, the control mice treated with normal mouse serum failed to inhibit the tumor growth. These results suggest the possible development of a tumor vaccine from the anti-idiotypic antibody (Ab2) prepared against the anti-BCG monoclonal antibody, for tumors sharing a common antigen(s) with Mycobacterium bovis strain BCG.

Oral cholera vaccines containing B-subunit-killed whole cells and killed whole cells only. I. Cross-reacting antigens of members of family Vibrionaceae and the vaccines.

Antigenic similarity between the oral cholera B subunit-whole cell (BS-WC) and whole-cell only (WC) vaccines and strains of the family Vibrionaceae was studied by crossed immunoelectrophoresis. A reference system consisting of Vibrio cholerae Inaba E1 Tor antigenic extract and homologous rabbit antiserum was applied in the study. The system was represented by 30 anodically migrating antigens forming distinct precipitation bands. Antigenic extracts of other members of the family Vibrionaceae showed the following numbers of cellular antigens shared in common with the reference system: Vibrio cholerae non-01-30, V. mimicus-23, V. fluvialis-15, V. parahaemolyticus-10, Aeromonas hydrophila-7, A. sobria-5, A. caviae-4 and Plesiomonas shigelloides-5 antigens. Homologous rabbit antiserum reacted with 11 antigens of BS-WC vaccine and 10 antigens of WC vaccine. The number of antigens which members of the family Vibrionaceae shared in common with those preserved in the WC component of the vaccines were as follows: Vibrio cholerae non-01, 7; V. mimicus, 5; V. fluvialis, 3; V. parahaemolyticus, 3; Aeromonas hydrophila, 2; A. sobria, 2; and A. caviae, 2; Plesiomonas shigelloides, 1. None of the strains produced an antigen reacting with anti-cholera toxin antibodies. The presence of common antigens in the vaccine and among members of family Vibrionaceae indicates that the oral cholera vaccine could stimulate immunity effectively against other members of the family.

Antigenic analysis between BCG and tumor cells by BCG-monoclonal antibodies.

Antigenic relation between Mycobacterium bovis strain BCG and experimental animal tumor cells was analyzed with BCG monoclonal antibodies (BCG-MoAbs). Four BCG-MoAbs of 602, 603, 609, and 612 were successfully established and applied for the antigenic analysis of Meth A, RL male 1, colon tumor 26 of mouse, and line 10 of guinea pig tumor. MoAb 602 showed broad reactivity against all types of tumor cells. BCG antigen(s) was clearly recognized as small granules on the tumor cell surface under the fluorescence microscope, indicating that the animal tumor cells shared the common antigen(s) with BCG.

Isolation of line 10 hepatoma-cell membrane by the water extraction method and immunochemical analysis.

Water extraction method was applied to isolate the cell membrane from line 10 hepatoma cells and normal liver cells in strain 2 guinea pig. The materials isolated by this method were further analyzed by different immunochemical techniques including SDS-PAGE, crossed immunoelectrophoresis and crossed affino immunoelectrophoresis to demonstrate the major components and their antigenicities. Five major glycoproteins of apparent molecular weights of 44, 46, 62, 64, and 68 kDa were prominent in line 10 tumor cell materials, whereas one band of molecular weight of 82 kDa was prominent in the materials from normal liver cells. Also four minor components from line 10 tumor cells were found to be glycoprotein in nature.

[Rapid diagnosis of experimental tumor by FITC-Con A lectin--a comparative study of smear and section specimens].

FITC-Con A staining as a rapid diagnostic method for tumor cells was applied to the tumors smeared on glass slide and section specimens to evaluate the reactivity with FITC-Con A. Good staining results were obtained in smear specimens with clear fluorescence on the membrane of tumor cells. Con A and LCH lectins bound well with tumor cells to produce strong fluorescence in comparison with PEA and DBA. It indicates that tumor cells expressed dominantly the receptors of alpha-D-glucose and alpha-D-mannose sugar chain on the membrane of tumor cells. From these results it was concluded that FITC-Con A staining method applied to smear specimens is more advantageous in the rapidity and the simplicity for tumor cell diagnosis than section specimen method.

Immunobiological relationships between Vibrio fluvialis and Vibrio cholerae enterotoxins.

A total of 26 strains of Vibrio fluvialis was included in this study, which were isolated from patients with diarrhoea and other sources. The GM1 enzyme linked immunosorbent assays performed with the culture filtrates of V. fluvialis yielded negative results, indicating that their receptor site is different from that of the known labile toxin. The cholera antitoxin failed to neutralize the skin permeability factor activities of all the V. fluvialis culture filtrates and none of the concentrated culture filtrates gave any precipitin band, when tested against the cholera antitoxin in Ouchterlony's gel diffusion test. These observations suggest that the toxin of V. fluvialis differs from the known cholera toxin in receptor site, mode of action and antigenicity.