Standard Reference Strains Research Articles

Molecular epidemiology of measles virus in Kenya

Measles causes significant morbidity and mortality globally. Many countries have embarked on immunization programs to control and prevent measles outbreaks and eventually to eliminate endemic measles. Kenya is currently in the outbreak control and prevention stage for measles. Measles virus genotyping is important for molecular epidemiological purposes, including the documentation of the elimination of endemic measles virus strains from a country, and mapping of transmission pathways. In this study, we collected clinical specimens from measles outbreak cases in 2002 in Kenya for measles virus genotyping. We were able to isolate and/or detect measles virus in 10 cases from 5 of the 8 provinces in Kenya. All these Kenyan measles strains were determined to be genotype D4 strains when compared to the standard World Health Organization-designated measles virus reference strains. Interestingly, the Kenyan D4 strains clustered into two distinct D4 subgroups. In addition, the inclusion of other published D4 measles strains in this analysis indicated that there are four distinct D4 clusterings, or subgroups: Montreal-like, India-like, Johannesburg-like, and Ethiopia-like. This is the first measles molecular epidemiology study in Kenya and establishes the current endemic measles strain as genotype D4. Importantly, this study shows that the Kenyan D4 strains are distinct from the B3 measles strain found in West Africa and the D4 strains reported in Ethiopia.

Nucleotide variation in Sabin type 2 poliovirus from an immunodeficient patient with poliomyelitis.

The molecular and antigenic properties of a Sabin-like type 2 poliovirus, isolated from the stool samples of a 2-year-old agammaglobulinaemic child who developed paralysis 1 year after receiving the third dose of oral poliovirus vaccine, were analysed. The virus revealed 0.88 % genome variation in the VP1 region compared with the standard reference strain, compatible with replication of the virus in the intestine over approximately 1 year. The typical mutations in the 5'NCR and VP1 associated with reversion to neurovirulence for Sabin type 2 poliovirus were found. Despite this, the virus was characterized by both PCR and ELISA tests as Sabin-like and showed temperature sensitivity and neurovirulence in transgenic mice typical of the Sabin type 2 vaccine strain. Gammaglobulin replacement therapy led rapidly to virus clearance, which, when combined with treatment with the antiviral drug pleconaril, stopped virus excretion; no further virus shedding occurred. This is the first case of poliomyelitis and long-term excretion from an immunodeficient patient to be reported in Italy through the active 'Acute Flaccid Paralysis' surveillance system.

New variants of Vibrio cholerae O1 biotype El Tor with attributes of the classical biotype from hospitalized patients with acute diarrhea in Bangladesh.

The sixth pandemic of cholera and, presumably, the earlier pandemics were caused by the classical biotype of Vibrio cholerae O1, which was progressively replaced by the El Tor biotype representing the seventh cholera pandemic. Although the classical biotype of V. cholerae O1 is extinct, even in southern Bangladesh, the last of the niches where this biotype prevailed, we have identified new varieties of V. cholerae O1, of the El Tor biotype with attributes of the classical biotype, from hospitalized patients with acute diarrhea in Bangladesh. Twenty-four strains of V. cholerae O1 isolated between 1991 and 1994 from hospitalized patients with acute diarrhea in Matlab, a rural area of Bangladesh, were examined for the phenotypic and genotypic traits that distinguish the two biotypes of V. cholerae O1. Standard reference strains of V. cholerae O1 belonging to the classical and El Tor biotypes were used as controls in all of the tests. The phenotypic traits commonly used to distinguish between the El Tor and classical biotypes, including polymyxin B sensitivity, chicken cell agglutination, type of tcpA and rstR genes, and restriction patterns of conserved rRNA genes (ribotypes), differentiated the 24 strains of toxigenic V. cholerae O1 into three types designated the Matlab types. Although all of the strains belonged to ribotypes that have been previously found among El Tor vibrios, type I strains had more traits of the classical biotype while type II and III strains appeared to be more like the El Tor biotype but had some classical biotype properties. These results suggest that, although the classical and El Tor biotypes have different lineages, there are possible naturally occurring genetic hybrids between the classical and El Tor biotypes that can cause cholera and thus provide new insight into the epidemiology of cholera in Bangladesh. Furthermore, the existence of such novel strains may have implications for the development of a cholera vaccine.

Diversity of nonpathogenic bacteria associated with seeds of single rice crop in Zhejiang Province

Nonpathogenic bacteria associated with rice seed are important natural resources as potential biocontrol agents (BCA). It is necessary to understand the diversity of nonpathogenic bacteria of rice seed in different rice ecosystems so as to utilize BCA for effective management of major diseases of rice. A survey on diversity of nonpathogenic bacteria was conducted in Zhejiang Province, China during 1996～2001. Six hundred and six rice seed samples were collected from plains, hills and mountainous areas. Over 9600 bacterial isolates were isolated and tested for colony morphology, pathogenicity, and some characteristics of bacteriology including Gram staining, fluorescent pigment and diffusible non fluorescent pigment on Kings medium B, production of yellow colony on YDC, aerobic character, and microscopic observation for endospore and flagellum. Together with 80 standard reference strains, 622 representative nopathogenic bacterial isolates were confirmed by Biolog and gas chromatographic analysis of fatty acid methyl esters and screened for BCA against the pathogens of sheath blight and bakanae disease of rice. Eleven bacterial species of Pseudomonas and 14 other genera of 23 species were identified, of which 55% of Pseudomonas species and 49% of other species were detected with antagonistic strains that varied with species. Bacillus spp., Acinetobacter spp., and P. putida are the major groups in the three rice growing areas where other similar bacterial species also exist. However, great variation was noted in isolation frequency of the seven common species in the areas.

Persistent infections and immunity in cystic fibrosis

Cystic fibrosis (CF) is the most common autosomal recessive lethal disease in the Caucasian population. Chronic respiratory infections with Pseudomonas aeruginosa, neutrophil-dominated airway inflammation and progressive lung damage are the major causes of morbidity and mortality in CF. Two persistent infection phenotypes expressed by this bacterium are biofilm and mucoidy. Biofilm, also called the microcolony mode of growth is the surface-associated adherent bacterial community, while mucoidy refers to a phenotype conducive to copious amounts of mucoid exopolysaccharide (MEP)/alginate that provides a matrix for mature biofilms conferring resistance to host defenses and antibiotics. Recent completion of the whole genomic sequence of the standard reference strain P. aeruginosa PAO1 has led to discoveries that many clinical isolates of this species possess unique genomic sequences (genomic islands) due to horizontal gene transfer. We propose this type of genetic exchange may play an important role in causing intrinsic genomic diversity of this organism. Therefore, the diversity, as revealed through profiles of restriction fragment length polymorphism (RFLP), may be linked to an array of novel and unexplored pathogenic mechanisms in P. aeruginosa. CF mouse models, while displaying many clinical similarities to human CF, have yet to demonstrate a chronic pulmonary disease phenotype. This review is intended to provide an overview of P. aeruginosa persistent infection phenotypes (biofilm and mucoidy) and an aerosol infection mouse model for CF. Genomic diversity of P. aeruginosa and its implications in the pathogenesis in CF will also be discussed.

Variation in susceptibility to Bacillus thuringiensis toxins among unselected strains of Plutella xylostella.

So far, the only insect that has evolved resistance in the field to Bacillus thuringiensis toxins is the diamondback moth (Plutella xylostella). Documentation and analysis of resistant strains rely on comparisons with laboratory strains that have not been exposed to B. thuringiensis toxins. Previously published reports show considerable variation among laboratories in responses of unselected laboratory strains to B. thuringiensis toxins. Because different laboratories have used different unselected strains, such variation could be caused by differences in bioassay methods among laboratories, genetic differences among unselected strains, or both. Here we tested three unselected strains against five B. thuringiensis toxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca, and Cry1Da) using two bioassay methods. Tests of the LAB-V strain from The Netherlands in different laboratories using different bioassay methods yielded only minor differences in results. In contrast, side-by-side comparisons revealed major genetic differences in susceptibility between strains. Compared with the LAB-V strain, the ROTH strain from England was 17- to 170-fold more susceptible to Cry1Aa and Cry1Ac, respectively, whereas the LAB-PS strain from Hawaii was 8-fold more susceptible to Cry1Ab and 13-fold more susceptible to Cry1Da and did not differ significantly from the LAB-V strain in response to Cry1Aa, Cry1Ac, or Cry1Ca. The relative potencies of toxins were similar among LAB-V, ROTH, and LAB-PS, with Cry1Ab and Cry1Ac being most toxic and Cry1Da being least toxic. Therefore, before choosing a standard reference strain upon which to base comparisons, it is highly advisable to perform an analysis of variation in susceptibility among field and laboratory populations.

In-vitro antimicrobial activity and chemical composition of Sardinian Thymus essential oils.

Essential oils and their components are becoming increasingly popular as naturally occurring antimicrobial agents. In this work the chemical composition and the antimicrobial properties of Thymus essential oils and of their main components were determined. Three essential oils obtained from different species of Thymus growing wild in Sardinia and a commercial sample of Thymus capitatus oil were analysed. The essential oil components were identified by GC/MS analysis. The antimicrobial activity of the oils and components was determined against a panel of standard reference strains and multiple strains of food-derived spoilage and pathogenic bacteria, using a broth microdilution method. The GC/MS analysis showed that the major constituents of the oils were monoterpene hydrocarbons and phenolic monoterpenes, but the concentration of these compounds varied greatly among the oils examined. The results of the antimicrobial assay showed that essential oils extracted from Sardinian Thymus species have an antimicrobial activity comparable to the one observed in other thyme oils. It seems also confirmed that the antimicrobial properties of thyme essential oils are mainly related to their high phenolic content. Among the single compounds tested carvacrol and thymol turned out to be the most efficient against both reference strains and food-derived bacteria. The results of this study confirmed the possibility of using thyme essential oils or some of their components in food systems to prevent the growth of foodborne bacteria and extend the shelf-life of processed foods.

Rapid DNA extraction methods and new primers for randomly amplified polymorphic DNA analysis of Giardia duodenalis

A randomly amplified polymorphic DNA (RAPD) procedure using simple genomic DNA preparation methods and newly designed primers was optimized for analyzing Giardia duodenalis strains. Genomic DNA was extracted from in vitro cultivated trophozoites by five freezing-thawing cycles or by sonic treatment. Compared to a conventional method involving proteinase K digestion and phenol extraction, both freezing-thawing and sonication were equally efficient, yet with the advantage of being much less time- and labor-intensive. Five of the 10 tested RAPD primers produced reproducible polymorphisms among five human origin G. duodenalis strains, and grouping of these strains based on RAPD profiles was in agreement among these primers. The consistent classification of two standard laboratory reference strains, Portland-1 and WB, in the same group confirmed previous results using other fingerprinting methods, indicating that the reported simple DNA extraction methods and the selected primers are useful in RAPD for molecular characterization of G. duodenalis strains.

Rapid detection of Mycobacterium bovis on its lipid profile by thin layer chromatography

Sixteen Mycobacterium bovis (M. bovis) strains isolated from bovine tissues and one standard reference strain of M. bovis AN5 alongwith other species of mycobacteria for comparison were investigated for the presence of phenolic glycolipid (PGL) and phthiocerol dimycocerosate (PDIM) for rapid identification of M. bovis by thin-layer chromatography (TLC). The study indicated presence of PGL with an Rf value of 0.75 in chloroform-methanol solvent in all 17 M. bovis strains. The dimycocerostate A corresponding to spot A was the major constituent among all the three spots in M. bovis strains. TLC appeared to be a promising alternative to conventional biochemical methods for identification of M. bovis taking into consideration both PGL and PDIM lipids.

Chromosome profile ofLeishmania donovani: Interstrain and interspecific variations

The genome ofLeishmania donovani AG83, a virulent strain causing kala-azar, was resolved into 29 chromosomal bands by pulsed field gel electrophoresis (pFGE) under standardized conditions. Comparison of the karyotype with those of other strains and species revealed variations. By Southern hybridization, specific genes were localized to individual chromosomes. Twenty-two copies ofβ-tubulin genes are located on band 27 (1.63 Mb); minor copies are present in band 16 (850 kb) and band 9 (650 kb). Aβ-tubulin related nontranscribed locus was isolated from a genomic library and shown to contain repetitive sequences hybridizing throughout the genome. Single chromosomes contain multicopy clusters of gp63 and rnini-exon-derived RNA genes, but interspecific variations were observed in each case. The results emphasize the importance of using a standard reference strain ofLeishmania donovani for coordinated genome mapping of this clinically important organism.

In Vitro and In Vivo Activities of Sparfloxacin in Legionella Infections

Legionella is a potential causative pathogen in pneumonia. It is also a facultative intracellular bac­ terium and, because it multiplies in macrophages, results of in vitro susceptibility tests do not always reflect clinical therapeutic effect. For this reason, it is necessary to test for both intracellular penetra­ tion of the agent and bactericidal activity against intracellular multiplying Legionella. In this study, the antibacterial activity of sparfloxacin, a newly developed fluoroquinolone, against Legionella was studied and compared with that of ofloxacin, ciprofloxacin, norfloxacin, and erythromycin. 1. Materials and Methods The minimum inhibitory concentrations (MICs) for the Legionella species were determined by the micro dilution broth method. A total of 68 Legionella strains were used in this study: standard reference strains of L. pneumophila (serogroups 1 to 13, n = 14) and Legionella spp. (n = 27); and clinical isolates of L. pneumophila (serogroup 1, n = 21; serogroup 2, n = 2; serogroup 3, n = 1; Table I. Minimum inhibitory concentration (MIG) of 5 antibacterial

Twelve aberrant strains of Staphylococcus aureus subsp. aureus from clinical specimens

A new biovar of Staphylococcus aureus subsp. aureus was isolated from human clinical specimens and described on the basis of studies of 12 isolates that were compared with 11 standard reference strains. Both DNA hybridization experiments and numerical taxonomy analysis demonstrated that these strains were strictly related to S. aureus subsp. aureus; however, they were significantly different from the latter. The atypical strains belonging to the new biovar can be distinguished from typical S. aureus subsp. aureus strains by their alpha-chymotrypsin, alpha-glucosidase, beta-N-acetylglucosaminidase, lipase (C-14), and leucine arylamidase enzymatic activities and novobiocin resistance. Thus, the combination of alpha-glucosidase and beta-N-acetyl-glucosaminidase is more useful for distinguishing these S. aureus strains from the other, typical ones.

Restriction Endonuclease Analysis of Delmarva Field Isolates of Infectious Laryngotracheitis Virus

Restriction endonuclease (RE) digestion patterns of six Delmarva field isolates of infectious laryngotracheitis virus (ILTV) were compared with three standard reference strains. With one exception, all of the field isolates generated RE digestion patterns identical to an embryo-propagated vaccine strain of ILTV when the six-base-recognizing REs EcoRI, HindIII, PstI, and BamHI were used. In order to increase the sensitivity of the RE analysis technique, a more sensitive DNA fingerprinting approach using four-base-recognizing enzymes was developed. One field isolate could be differentiated from the embryo-propagated vaccine strain using all three enzymes, Sau3AI, MspI, and HinfI. A second isolate could be differentiated only by comparing HinfI digestion patterns. This work provides additional evidence that differentiable strains of ILTV exist in the United States. Furthermore, currently used RE analysis methods may not be sensitive enough to discriminate between field isolates and vaccine strains of ILTV, thus challenging the theory that vaccine strains of ILTV are responsible for field outbreaks of infectious laryngotracheitis.

Permeability of the outer membrane of Moraxella catarrhalis for β-lactam antibiotics

The susceptibility of ten clinical isolates and a standard reference strain, ATCC25238, of Moraxella catarrhalis to 22 beta-lactam antibiotics was examined and compared with that of Escherichia coli strain B. All the strains of M. catarrhalis tested, especially the non beta-lactamase-producing strain ATCC25238, were more susceptible to a rang of structurally unrelated beta-lactam antibiotics, including small Mr carbapenems, than E. coli B. The permeability of the M. catarrhalis outer membrane to beta-lactam antibiotics was examined by the swelling technique with proteoliposomes reconstituted from outer membranes. The diffusion rate of beta-lactams through the liposome membrane was inversely related to their Mr, a relationship which might be expected for entry by diffusion through a porin.

Resistance risk assessment of two insect development inhibitors, diflubenzuron and cyromazine, for control of the housefly Musca domestica L. Part II: Effect of selection pressure in laboratory and field populations

AbstractLaboratory selection with diflubenzuron (DFB) and cyromazine (CYR) was carried out with strains of houseflies collected on Danish farms. The adult flies were multiresistant to organophosphorus, carbamate and pyrethroid insecticides. Susceptibility to the larvicidal effect of DFB or CYR was monitored on Danish farms where fly control with DFB or CYR had been carried out for one to nine years. On these farms the fly population continued during the winter, but at a low level.Four strains were selected with DFB in seven out of 13–18 laboratory generations. The selections varied from 56% to 94% mortality. The strains showed 1–5 times increase of LC50 and LC95 to DFB. The larvicidal effect of DFB was monitored on five farms where treatment of fly breeding sources in manure (1 g DFB m−2) was carried out regularly for one to nine fly seasons. On none of the farms was any general increase of tolerance to DFB found. Resistance ratios (R/S) compared to the WHO Standard Reference Strain ranged from 0.8 to 2.0 at LC50 and from 1–0 to 2–9 at LC95.Two strains were selected with CYR in five or six out of 13 laboratory generations. The selections varied from 62 to 97% mortality. The strains showed no increase of LC50 or LC95. The larvicidal effect of CYR was monitored on three farms where housefly breeding places in the animal houses were treated with CYR (0.5 g m−2) for two fly seasons. No reduction of susceptibility to CYR was found and the R/S was 0.7 to 0.9.The effect of DFB or CYR selection pressure is compared with other investigations and discussed. Moderate to high resistance to DFB or CYR can develop in housefly populations if the selection pressure is strong, especially when used as feed‐through applications on poultry farms where all feed contains DFB or CYR. If the treatment of fly breeding sources is less complete, resistance problems may not develop.

Isolation of Mycoplasma synoviae from the Brains of Commercial Meat Turkeys with Meningeal Vasculitis

Mycoplasma synoviae (MS) was isolated from the brains of 22-week-old commercial meat turkeys displaying severe synovitis and infrequent central nervous system signs. Histological examination of the brains revealed mild-to-severe meningeal vasculitis. The vasculitis ranged from fibrinoid necrosis with little inflammation to a marked infiltration of lymphocytes and plasma cells disrupting the architecture of the vessel wall, accumulating as perivascular cuffs, and involving surrounding meninges. Occasional arteries were undergoing thrombosis. Similar lesions were occasionally seen in renal, synovial, and splenic vessels. MS isolates from the brain, trachea, and joint showed similar protein-banding patterns by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, the protein profile differed markedly from the standard MS reference strain, WVU 1853. This is the first known field case of MS isolation from the brains of turkeys.

Efficacy of NY-198 against experimental Legionnaires disease.

The in vitro and in vivo effects of NY-198 against Legionella pneumophila were compared with those of ciprofloxacin. The MIC of NY-198 against 15 standard reference strains of Legionella of various species, between 0.03 and 0.125 micrograms/ml, was the same as that of ciprofloxacin. The peak concentration of NY-198 in the lungs and sera of guinea pigs with experimentally induced Legionella pneumonia was higher than that of ciprofloxacin after oral administration. The overall survival rate was higher in animals treated with NY-198 than in those treated with ciprofloxacin. Thus, NY-198 appears valuable in the treatment of Legionnaires disease.

Characteristics and Adaptability of Some New Isolates of Clostridium thermocellum

Six strains of Clostridium thermocellum isolated from various environments were characterized as to growth rate, production of reducing sugars, ethanol, and acetic acid from cellulose, base composition of DNA, and the abilities to adapt to ethanol and to grow at 45 degrees C. Five of the six new isolates produced 7 to 15% more ethanol and two produced about 45% more reducing sugars than a standard reference strain. One strain (MC-6) adapted more readily to growth in 2% ethanol than the others.

Contribution of the Symbiotic Plasmid to the Competitiveness of Rhizobium leguminosarum

Four different symbiotic plasmids from Rhizobium leguminosarum were introduced into three different recipient strains that lacked plasmid-linked symbiotic determinants. The twelve synthetic strains so constructed were each tested for competitiveness against a standard reference strain. The recipient strain and the introduced symbiotic plasmid contributed about equally to competitiveness in forming root nodules on pea plants: there was also significant interaction between strain and plasmid, although this was much less important than the main effects. Competitiveness for growth on the legume root surface (the rhizosphere) was attributable entirely to the recipient strain; the introduced plasmid had no significant effect.

A clinico-pathological study of a case of kuru

Kuru was diagnosed in a 42-year-old Melanesian male from the Eastern Highlands Province of Papua New Guinea. The clinical features indicated predominant cerebellar degeneration together with widespread cortical neuronal dysfunction and involvement of the diencephalon, hippocampus and basal ganglia. Dementia was an early and prominent feature. The duration of clinical illness was about 12 months and atypically he spent the last 7 months in hospital allowing continuous assessment. At autopsy, spongiform encephalopathy was demonstrated, and inoculation of brain tissue into 4 squirrel monkeys and 1 capuchin monkey resulted in the development of kuru. This is the longest continuous study of kuru in a hospital setting to be recorded in the adult human subject. The virus isolated from the brain of this patient has been adopted as a standard reference strain of kuru for future use and repository.