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Volume 50,
Issue 8,
2001
Volume 50, Issue 8, 2001
- Editorial
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- Diagnostic Microbiology
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A comparison of the isolation rates of Salmonella and thermophilic Campylobacter species after direct inoculation of media with a dilute faecal suspension and undiluted faecal material
More LessRegardless of media used, dilution of faecal samples before direct plating may improve isolation rates and reduce subcultures by freeing organisms from the faecal mass and diminishing competing flora. Despite the routine use of dilution in many laboratories, it has never been established properly whether direct or dilute inocula should be used in primary plating of faeces. A total of 3764 faecal samples was examined in four laboratories with a standardised methodology. The isolation rates, competing flora and confirmatory work performed for Salmonella spp. and Campylobacter spp. from primary plating media with a dilute faecal inoculum were compared with those after direct inoculation of faecal material. Inoculum effects on the isolation of Shigella spp. could not be assessed as only one isolate occurred during the study period. The overall isolation rates of both major enteric pathogens were unaffected by the inoculum. However, significantly fewer wasted subcultures were recorded with a dilute inoculum for Campylobacter spp., and competing flora was reduced in all cases without diluting out small numbers of the pathogen.
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Evaluation of three methods to measure anti-Brucella IgM antibodies and interference of IgA in the interpretation of mercaptan-based tests
More LessThe results of a dipstick assay for the detection of immunoglobulin M (IgM) to Brucella smooth lipopolysaccharide (S-LPS) correlated with those of an enzyme-linked immunosorbent assay (ELISA) for IgM and of the serum agglutination test (SAT) performed with and without dithiothreitol. Two sera which were dithiothreitol-sensitive and were dipstick negative were shown to contain specific IgA. The dipstick assay is recommended as a simple method for detecting specific IgM antibodies in acute-phase brucellosis patients.
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Sensitivity and performance characteristics of a direct PCR with stool samples in comparison to conventional techniques for diagnosis of Shigella and enteroinvasive Escherichia coli infection in children with acute diarrhoea in Calcutta, India
More LessAs the sensitivity of the conventional techniques for identifying Shigella spp. and enteroinvasive Escherichia coli (EIEC) causing dysentery cases is low, a PCR assay was evaluated in this study. Analytical sensitivity (2 × 102 cfu) of the PCR technique was obtained by artificially spiking negative stool samples with a standard strain of S. flexneri type 2, then determining the detection limit. Specificity (100%) of the method was determined by testing a number of known Shigella and EIEC strains and organisms other than Shigella spp. A total of 300 stool samples collected from children with acute diarrhoea was plated on to two selective agar media after enrichment in Luria broth. Shigella spp. were isolated from 7.7% (23 of 300) and EIEC from 1% (3 of 300) patients. All enriched stool samples were subjected to PCR to amplify the target sequence of invasive plasmid antigen (ipa)H locus, a multicopy element found on the chromosome and invasion plasmid. The stool PCR was positive in 24 of the 26 culture-positive and in 22 culture-negative stools, thus detecting the presence of Shigella spp. or EIEC in 15.3% (46 of 300) of diarrhoea cases. When an ial probe was used for colony hybridistion with enriched stool cultures blotted on to membranes, 9.6% (29 of 300) of dysentery cases were identified as being caused by Shigella spp. or EIEC. Thus the sensitivity of enriched stool culture, colony hybridisation and enriched stool PCR was found to be 54%, 60% and 96%, respectively, when each of the methods was compared to the total microbiologically confirmed cases of dysentery. It was also observed that only 38% (48 of 126) of acute bloody dysentery cases actually had shigella or EIEC infection, as confirmed by laboratory methods. Moreover, this PCR assay could identify a number of untypable Shigella strains (Sh OUT), which would have remained undiagnosed had this assay not been used.
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- Antimicrobial Agents And Resistance
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Identification of catalase-like activity from Mycobacterium leprae and the relationship between catalase and isonicotinic acid hydrazide (INH)
More LessAs Mycobacterium leprae proliferate inside macrophages, it has been speculated that catalase encoded by katG may protect the bacilli from deleterious effects of peroxide generated from the macrophage and may also play a crucial role in the survival of M. leprae in vivo. However, unlike that of M. tuberculosis, the katG of M. leprae has been reported to be a pseudogene, implicating that isoniazid, which is activated to a potent tuberculocidal agent by catalase, is unlikely to be of therapeutic benefit to leprosy patients. These results raise a question as to how M. leprae avoids H2O2-mediated killing inside macrophages. To understand the survival of M. leprae in macrophages, the present study attempted to detect catalase-like activity in M. leprae. Catalase-like activity was found in M. leprae cell lysate by the diaminobenzidine (DAB) staining method with non-denaturing polyacrylamide gel electrophoresis. An ammonium sulphate precipitation study revealed that the catalase-like activity was precipitable with 80% ammonium sulphate. The effect of isoniazid (INH) on M. leprae growth was also tested by RT-PCR and radiorespirometric assay to examine catalase-like activity in M. leprae, because INH was activated by catalase. It was found that the viability of M. leprae was decreased at a concentration of 205 μg/ml by radiorespirometric assay and it was inhibited at higher concentrations as determined by RT-PCR. These data suggest that a catalase-like activity other than that encoded by katG is present in M. leprae.
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Characterisation of VanA and VanB elements from glycopeptide-resistant Enterococcus faecium from Greece
More LessTen glycopeptide-resistant Enterococcus faecium isolates from separate patients in Laikon General Hospital, Athens were studied. Eight isolates had the VanA phenotype and represented variants of three strains based on SmaI macrorestriction banding patterns. Their VanA elements were compared with the prototype element, Tn1546, by an overlapping PCR method. Three related isolates contained resistance elements indistinguishable from Tn1546 (designated Greek type I). The other five isolates all contained identical elements that differed from Tn1546 by the presence of IS1251 between vanS and vanH, by a point mutation (G ← T) at nucleotide position 8234 within vanX and by a partial loss of transposition gene orf1 (designated Greek type II). Two distinct strains of E. Caecium with the VanB phenotype were obtained. HhaI digestion of an amplified fragment of the vanB gene indicated that both strains contained the vanB2 allele, and further PCR assays confirmed that the vanB2 gene cluster was located within a Tn5382-like element.
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- Bacterial Epidemiology And Typing
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Colonisation and transmission of Clostridium difficile in healthy individuals examined by PCR ribotyping and pulsed-field gel electrophoresis
Healthy adults who had not been exposed to antimicrobial agents for the preceding 4 weeks were examined for intestinal carriage of Clostridium difficile. The 1234 individuals examined were composed of seven groups: three classes of university students, hospital workers at two hospitals, employees of a company and self-defence force personnel at a local station. Overall, 94 (7.6%) individuals were positive for C. difficile by faecal culture but carriage rates among the study groups ranged from 4.2% to 15.3%. Typing by PCR ribotyping and pulsed-field gel electrophoresis demonstrated clusters of carriers colonised by a single type in each of three groups, indicating that cross-transmission of C. difficile can occur in community settings. Follow-up culture was performed on 38 C. difficile-positive individuals and C. difficile was isolated again from 12 (32%) of them 5–7 months after the initial culture; six (50%) of these 12 individuals had a new strain on repeat culture. Two or more family members were C. difficile-positive in five of 22 families examined. C. difficile with an identical type was isolated from persons within a family in only one family. These results suggest that intestinal carriage by healthy adults may play a role as a reservoir for community-acquired C. difficile-associated diarrhoea, but that cross-transmission of C. difficile does not occur frequently among family members at home.
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Flagellin gene PCR-RFLP analysis of a panel of strains from the Burkholderia cepacia complex
More LessBurkholderia cepacia, an important opportunist pathogen, is genetically heterogeneous. The B. cepacia complex has been subdivided into a number of genospecies or genomovars. A flagellin gene PCR-RFLP method was applied to a representative panel of strains of known genomovar. The technique was able to distinguish strains of B. multivorans from other members of the B. cepacia complex on the basis of amplicon size (typical of type I rather than type II flagellins) with the exception of one genomovar I strain. There was considerable variation in RFLP patterns amongst the panel of strains; only two pairs of strains were indistinguishable with both HaeIII and MspI digestion. Where RFLP patterns matched with both enzymes or a single enzyme, matching strains were always in the same genomovar. It was possible to distinguish the UK cystic fibrosis epidemic strain from all other members of the panel, including nine other genomovar III strains. The level of variation suggests that flagellin genotyping is a useful method for discriminating between B. cepacia strains.
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Analysis of different molecular methods for typing methicillin-resistant Staphylococcus aureus isolates belonging to the Brazilian epidemic clone
The extensive geographic spread of MRSA isolates belonging to the Brazilian epidemic clone (BEC) limited the value of pulsed-field gel electrophoresis (PFGE) in epidemiological studies of outbreaks caused by these strains. Thus, the discriminatory power of eight different molecular methods was evaluated in an attempt to establish a methodology for genotyping BEC isolates involved in intra-hospital outbreaks. BEC isolates from five hospitals in Teresina City, Piauí State were genotyped by conventional electrophoresis or PFGE of ClaI- or SmaI-digested genomic DNA hybridised with specific labelled mecA, Tn554, IS257 and IS256 probes. The combination of PFGE with ClaI/mecA, ClaI/Tn554, ClaI/IS257, SmaI/mecA and SmaI/IS257 probe-fingerprinting techniques provided a very poor discriminatory power for BEC strains. Although ClaI/IS256 fingerprinting discriminated 17 different polymorphisms among the isolates displaying PFGE A1 pattern, this strategy was not reproducible. In contrast, the combination of PFGE and SmaI/IS256 polymorphisms differentiated BEC isolates into nine stable polymorphisms. Thus combination of PFGE and hybridisation with IS256 probe may be recommended as a useful means of typing BEC strains involved in intra-hospital infections.
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- Microbial Pathogenicity
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Role of T cells in granuloma formation induced by Rhodococcus aurantiacus is independent of their interferon-gamma production
More LessIntravenous injection of Rhodococcus aurantiacus into mice causes granulomatous inflammation dependent on endogenous interferon-γ (IFN-γ). This study investigated the mechanism of granuloma formation with an adoptive transfer system in IFN-γ knockout (IFN-γ−/−) mice. IFN-γ−/− mice infected with R. aurantiacus did not develop granulomas, and high titres of endogenous interleukin-10 (IL-10) were detected in spleen extracts at 2 weeks after infection. The adoptive transfer of splenocytes from infected wild-type (IFN-γ+/+) mice did not restore granuloma formation, although this treatment diminished IL-10 production in IFN-γ−/− mice. Adoptive transfer of splenocytes from infected IFN-γ−/− mice into infected IFN-γ+/+ reduced granuloma formation. These results suggest that splenocytes of IFN-γ−/− mice suppress granuloma formation. On the other hand, although IFN-γ production induced by R. aurantiacus infection was detected in nude mice, which are deficient in T cells, granuloma formation was not induced in them. However, adoptive transfer of immune splenocytes from either IFN-γ+/+ mice or IFN-γ−/− mice could induce granuloma formation. This means that splenocytes of IFN-γ−/− mice have the ability to both induce and suppress granuloma formation. Induction of granuloma is probably dependent on both T cells and IFN-γ produced by non-T cells. It is suggested that the role of T cells in granuloma formation is not dependent on their IFN-γ production.
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Development of systemic bacteraemia after oral inoculation of vancomycin-resistant enterococci in mice
Bacteraemia caused by vancomycin-resistant enterococci (VRE) is an important clinical problem because there are only a few potent antimicrobial agents against such bacteria. Therefore, understanding the pathogenic mechanisms of VRE bacteraemia is important for prophylaxis. This study shows that treatment of mice with cyclophosphamide and a combination of metronidazole, kanamycin and vancomycin reduced normal intestinal flora and induced systemic VRE bacteraemia. Translocation of VRE and the normal intestinal flora to the mesenteric lymph nodes, liver, spleen and blood, and mortality rate were dependent on treatment with cyclophosphamide and each of the three antimicrobial drugs. Among the different strains studied, C57BL/6 mice were the most susceptible to VRE. The virulence of vancomycin-resistant Enterococcus faecalis was greater than that of vancomycin-resistant Ent. faecium. On the day after inoculation of VRE, Escherichia coli was also detected in many VRE-positive specimens including blood, liver and the mesenteric lymph nodes. Moreover, both VRE and E. coli were detected simultaneously in almost all blood samples obtained from dead and dying mice, and VRE organisms outnumbered E. coli in those samples by 100:1 or more. These results indicate that changes in normal intestinal flora by administration of antimicrobial drugs and severity of neutropenia induced by cyclophosphamide are important factors that contribute to the development of systemic VRE bacteraemia. E. coli may be intimately associated with the establishment of VRE translocation.
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Susceptibility of irradiated mice to Bacillus anthracis Sterne by the intratracheal route of infection
The susceptibility of sublethally irradiated mice to pulmonary infection with Bacillus anthracis was investigated in a mouse model. Female B6D2F1/J mice were challenged intratracheally with 4.3×106, 3.7×107 and 4.4×108 cfu of B. anthracis Sterne spores 4 days after 60Co γ-radiation at a dose of 0, 1, 2, 3, 4, 5, 6 or 7 Gy. Bacterial cultures were obtained from lung, spleen homogenates and heart blood. A biphasic mode of mortality was observed, with a constant response of up to 3 or 4 Gy (up to 18% mortality), after which a sharp increase in mortality occurred (up to 100%). When irradiation was delayed beyond 15 days after inoculation, the susceptibility to B. anthracis infection and subsequent mortality disappeared. B. anthracis was recovered from the organs and blood of up to 89% of the animals. However, organisms of enteric origin were also isolated mixed with B. anthracis from up to 36% of the animals exposed to 3, 5 or 7 Gy. Inoculation of B. anthracis Δ-Sterne-1 that lacks lethal toxin and oedema toxin also induced infection with B. anthracis, but not translocation of enteric micro-organisms. The synergic adverse effect of exposure to γ-radiation followed by intratracheal challenge with B. anthracis was observed above 4 Gy. The lethal toxin of B. anthracis may enhance the emergence of polymicrobial infection with B. anthracis and enteric micro-organisms.
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The N-terminal of thrombospondin-1 is essential for coagulase-negative staphylococcal binding
More LessBacterial binding was studied to determine whether thrombospondin-1 (TSP) acts as a ligand in attachment of coagulase-negative staphylococci (CNS). Twenty-five of 27 CNS strains bound soluble TSP. Staphylococcus epidermidis J9P bound 125I-labelled TSP in a dose-dependent manner. Scatchard plot analysis of the binding of TSP by strain J9P revealed two K d values of 6.4×10−95mum and 2.9×10−85mum. The binding structures of strain J9P were sensitive to protease and were resistant to heat treatment. Unlabelled TSP and recombinant von Willebrand factor inhibited binding of TSP by strain J9P, but other proteins or monosaccharides did not. Heparin inhibited binding of TSP to strain J9P and two other S. epidermidis strains, BD5703 and BD969. Fusion proteins of the type 1 repeats, type 2 repeats, type 3 repeats and C-terminal domain of TSP or the synthetic Arg-Gly-Asp peptide did not inhibit binding of TSP to bacteria. TSP promoted adhesion of S. epidermidis strains when it was immobilised on polymer surfaces. These results indicate that the specific interaction between CNS and TSP may contribute to bacterial adhesion on biomaterial surfaces. The N-terminal heparin-binding domain of TSP appears to be the major region for recognition by CNS.
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- Mycology
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Different isoforms of secreted aspartyl proteinases (Sap) are expressed by Candida albicans during oral and cutaneous candidosis in vivo
More LessDistinct isoforms of secreted aspartyl proteinases (Sap) of Candida albicans are important virulence factors for different types of candidosis. Predominant expression of Sap1–3 has been shown to be crucial for superficial infections in experimental mucosal and cutaneous candidosis, whereas Sap4–6 might be important for systemic disease. This in-vivo study investigated Sap expression in two samples from patients with oral candidosis and from cutaneous infection. Two different polyclonal antibodies directed against Sap1–3 and Sap4–6 were used for ultrastructural characterisation of protein localisation and expression. Post-embedding immuno-electron microscopy revealed Sap1–3 and Sap4–6 immunoreactivity in all samples. All C. albicans cells expressed predominantly the proteinases Sap1–3 which were evenly distributed within the cell wall and cytoplasmic membrane. In contrast, Sap4–6 labelling was only evident in a few fungal cells. In particular it was localised at the tips of hyphal cells during invasion. These data suggest a different pathogenetic role for Sap1–3 and Sap4–6 during host–fungal interaction.
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Volumes and issues
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Volume 74 (2025)
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Volume 73 (2024)
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Volume 72 (2023 - 2024)
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Volume 71 (2022)
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Volume 70 (2021)
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Volume 69 (2020)
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Volume 68 (2019)
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Volume 67 (2018)
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Volume 66 (2017)
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Volume 65 (2016)
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Volume 64 (2015)
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Volume 63 (2014)
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Volume 62 (2013)
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Volume 61 (2012)
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Volume 60 (2011)
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Volume 59 (2010)
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Volume 58 (2009)
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Volume 57 (2008)
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Volume 56 (2007)
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Volume 54 (2005)
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Volume 53 (2004)
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Volume 51 (2002)
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Volume 50 (2001)
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Volume 49 (2000)
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Volume 48 (1999)
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Volume 47 (1998)
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Volume 46 (1997)
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Volume 45 (1996)
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Volume 44 (1996)
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Volume 43 (1995)
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Volume 42 (1995)
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Volume 41 (1994)
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Volume 39 (1993)
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Volume 37 (1992)
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Volume 36 (1992)
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Volume 35 (1991)
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Volume 34 (1991)
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Volume 33 (1990)
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Volume 30 (1989)
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Volume 29 (1989)
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Volume 28 (1989)
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Volume 27 (1988)
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Volume 26 (1988)
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Volume 25 (1988)
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Volume 23 (1987)
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Volume 19 (1985)
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Volume 18 (1984)
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Volume 17 (1984)
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Volume 16 (1983)
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Volume 15 (1982)
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Volume 14 (1981)
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Volume 12 (1979)
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Volume 10 (1977)
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Volume 9 (1976)
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Volume 8 (1975)
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Volume 7 (1974)
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Volume 6 (1973)
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Volume 5 (1972)
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Volume 4 (1971)
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Volume 3 (1970)
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Volume 2 (1969)
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Volume 1 (1968)
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