Current Status of Immunofluorescence Techniques for Rapid Detection of Shigellae in Fecal Specimens

Polyvalent Shigella conjugates were prepared for Shigella groups A, B, C, and D. After preliminary testing with pure cultures of both homologous and heterologous organisms, these reagents were used in three evaluation studies. Fecal specimens from patients hospitalized with diarrhea, from children involved in an institutional outbreak of dysentery due to S. sonnei, and from patients with diarrhea in Arizona were screened by fluorescent-antibody (FA) tests and were cultured. Specimens were examined at various periods of time after collection and after incubation in broth and saline. Results showed that shigellae were detected most frequently when specimens were cultured immediately after collection. FA tests revealed more positive results when the specimens were incubated in either saline or broth than when they were examined immediately after collection. The S. sonnei conjugate gave the most reliable results of any of the Shigella FA reagents used in these investigations. It proved to be both sensitive and specific.     

Effects of caffeic acid, chlorogenic acid and ferulic acid on growth and arylamine N-acetyltransferase activity in Shigella sonnei (group D)

Arylamine N-acetyltransferase (NAT) activities with 2-aminofluorene (2-AF) as substrates were determined in Shigella sonnei (group D) collected from patients with diarrhoeal disease. The NAT activity was determined using an acetyl CoA recycling assay and high pressure liquid chromatography. Inhibition of growth studies from S. sonnei (group D) demonstrated that caffeic acid (CA), chlorogenic acid (CGA) and ferulic acid (FA) elicited a dose-dependent bactericidal effect in S. sonnei (group D) cultures, i.e. the greater the concentration of CA, CGA and FA, the greater the inhibition of growth of S. sonnei (group D). Cytosols or suspensions of S. sonnei (group D) with and without selected concentrations of CA, CGA and FA co-treatment showed different percentages of 2-AF acetylation. The data indicated that there was reduced NAT activity associated with increased CA, CGA and FA in Shigella dysenteriae (group D) cytosols and intact cells. For the cytosol and intact bacteria examinations, the apparent values of K(m) and Vmax decreased after being co-treated with 400 microM CA, CGA and FA. This report is the first demonstration of plant phenolic inhibition (CA, CGA and FA) of arylamine NAT activity and growth in the bacterium S. sonnei (group D).     

Preparation and Testing of Polyvalent Conjugates for Fluorescent-Antibody Detection of Salmonellae

A polyvalent OH conjugate for Salmonella O groups A through I, K, L, and O was prepared and tested against pure cultures of salmonellae, nonsalmonellae, and a variety of food, fecal, and environmental specimens. Examination of pure cultures revealed that the conjugate gave negligible staining with representative strains of Shigella, Proteus, Providence, Serratia, and Pseudomonas. However, it stained 12% of the Escherichia coli and Citrobacter freundii strains and 36% of the Arizona strains. Over 1,200 specimens of various types were examined by both fluorescent-antibody (FA) and cultural procedures. Results indicate that, when used with discretion, FA screening can be a useful tool for rapid presumptive indication of the presence of salmonellae. The need for careful selection of strains used for preparing antisera and the importance of adequate evaluation of Salmonella FA reagents are discussed.     

Rapid Identification and Typing of Herpes Simplex Virus Types 1 and 2 by a Direct Immunofluorescence Technique

An immunofluorescence (FA) technique has been developed which can identify herpes simplex virus (HSV) in clinical specimens and also type the virus directly as type 1 or type 2. This test, first applied to cervicovaginal specimens obtained from 80 mice genitally inoculated with HSV, indicated a sensitivity approaching 80% in comparison to standard viral isolation methods. A similar sensitivity was found when the test was applied to 185 clinical specimens with adequate cells for staining, which were obtained from a variety of sites of patients with suspect herpetic infection. In only 1 of 6 specimens positive by both FA and culture methods was the HSV type wrongly identified by the FA technique. There were also six specimens which were negative by culture methods but positive by the FA test, indicating a specificity of 91%. It is likely that these are not instances of false-positive tests but of other factors which may have resulted in negative viral isolations by culture methods. As more specific reagents become available, it is anticipated that the FA technique will have wider usage in diagnostic laboratories for the identification and typing of HSV types 1 and 2.     

Validity of Spinal Cord Examination as a Substitute Procedure for Routine Rabies Diagnosis

A significant portion of specimens received by this laboratory for rabies diagnosis is unsatisfactory for testing due to decomposition of the brain, or severe mutilation of the head when the animal was killed. Examination of the spinal cord was therefore explored as a possible alternative method when standard brain examination was not possible. In this study, both brain and spinal cord of 248 rabies-suspect animals were examined to assess the reliability of the spinal cord method. Brain and spinal cord of the 248 animals were examined by fluorescent antibody (FA) method, and mouse inoculation tests were performed on 247 brain specimens and 13 spinal cord specimens. By using both brain and spinal cord, 30 animals representing 8 species were diagnosed as rabid by FA, and 218, representing 11 species, were negative. There was 100% agreement between two procedures with FA as the criterion. This study showed that in cases where the usual examination is precluded due to brain destruction, the spinal cord procedure offers an equally reliable alternate method of diagnosis.     

Spotted fever group rickettsiae in immature and adult ticks (Acari: Ixodidae) from a focus of Rocky Mountain spotted fever in Connecticut

Immature and adult ixodid ticks were collected during 1983 and 1984 in Newtown, Connecticut, an area endemic for Rocky Mountain spotted fever (RMSF), to determine prevalence of infection by spotted fever group (SFG) rickettsiae. Direct fluorescent-antibody (FA) staining revealed SFG organisms in 6 (1.8%) of 332 Dermacentor variabilis larvae, 5 (7.8%) of 64 D. variabilis nymphs, and in 2 (40%) of 5 Ixodes cookei nymphs removed from small- and medium-sized mammals. Hemolymph tests detected rickettsia-like organisms in 15 (8.8%) of 170 D. variabilis adults; 8 specimens retested by direct FA were negative. In contrast, hemocytes from 5 (8.6%) of 58 Ixodes texanus females contained organisms that stained positively in both hemolymph and direct FA tests. An indirect microimmunofluorescence test identified specific antibodies to Rickettsia rickettsii, the etiologic agent of RMSF, in serum samples from a chipmunk, raccoons, and white-footed mice. Results indicate that immature or adult ticks of at least three species may be involved in the maintenance and transmission of SFG rickettsiae at Newtown.     

Multi-Biochemical Test System for Distinguishing Enteric and Other Gram-Negative Bacilli

A multi-biochemical test system consisting of nine tests, entitled Enterotube, was evaluated in parallel with conventional tests to determine its value in the identification of enteric and certain other gram-negative bacilli. The 242 bacterial strains studied were from a variety of pathological specimens and from our culture collection. When the results with individual tests represented in both test systems were compared, no discrepancies were noted in the indole test, and one discrepancy was recorded for dextrose. In 7 of 242 hydrogen sulfide tests, 3 of 242 phenylalanine tests, 22 of 242 urease tests, 15 of 242 dulcitol tests, 12 of 242 lactose tests, 27 of 217 lysine decarboxylase tests, and 5 of 242 citrate tests, the Enterotube results were contrary to those obtained with conventional methods. The lysine decarboxylase test in the Enterotube posed a problem of interpretation and readability and is not an acceptable alternative to the conventional methods. Fifteen of the strains studied were incorrectly identified by the Enterotube system and four could not be differentiated from other closely related strains. Salmonella could be identified as to group, whereas Shigella strains were frequently misidentified as Escherichia. The Enterotube method is simple and convenient, and all media are inoculated at once from a single colony.     

Fluorescent-Antibody Techniques for the Identification of Group D Streptococci: Direct Staining Method

Fluorescent-antibody (FA) techniques were employed in an attempt to develop a rapid test for the identification of group D streptococci. Fresh isolates were obtained from sewege and feces of sheep, cattle, horses, rabbits, chickens, geese, and rats. Identification to species were made by the conventional physiological, biochemical, and serological tests. Both whole and disrupted cells of representative strains of each species were used for the preparation of the group D streptococcus vaccine. Globulin fractions of individual and pooled antisera were labeled with fluorescein isothiocyanate, and the resulting conjugates were tested with homologous and heterologous antigens. The specificity of the conjugates and staining was assessed by adsorption and inhibition tests utilizing controls with homologous and heterologous antigens. Employing the direct staining method and individual and pooled conjugates, it was possible to obtain 84 and 85% positive FA reactions, respectively, with group D streptococcal strains. Trypsinization of the smears prior to staining eliminated all FA cross-reactions observed with non-group D streptococci and staphylococci. These findings suggest that the direct staining method will be of value in the rapid identification of group D streptococci.     

Direct Immunofluorescence Test for the Diagnosis of Genital Herpesvirus Infections

Herpetic lesions of the genitalia may be confused clinically with other ulcerative, genital lesions. Direct immunofluorescence (FA) provides a rapid method of diagnosis, and the utility of this method for the diagnosis of genital ulcers was examined. One hundred and ten patients with genital lesions were examined by darkfield for syphilis and by FA and culture for herpes simplex virus (HSV) infections. Satisfactory samples were obtained from 102 patients, of which 81 were clinically suspected cases of HSV. Acetone-fixed slides of scrapings of ulcerative lesions were stained with conjugated antiserum prepared in rabbits against HSV type 2. HSV was isolated from 73% of specimens of suspected herpetic lesions, and 77% of these specimens were positive by FA. Nine percent were positive by FA only and these were not thought to represent false positives. Five percent were positive by culture only. A comparison of clinical diagnoses with laboratory findings revealed that 4% of the cases were misdiagnosed when only the clinical evaluation was considered. The data suggest that the inclusion of a diagnostic FA test for HSV along with the darkfield examination may be useful for differentiating the etiological agents of ulcerative, genital lesions.     

Control of Shigella flexneri in Celebes black macaques (Macaca nigra)

Stool specimens collected systematically from a group of Celebes black macaques (Macaca nigra) with a high incidence of diarrhea were examined microbiologically. Numerous isolates of Shigella flexneri, Campylobacter jejuni and pathogenic Escherichia coli were recovered. Previous parasitology reports had revealed that the majority of the animals had Balantidium coli. Subsequently, the group was treated with trimethoprim-sulfamethoxazole, erythromycin and tetracycline. After treatment, Shigella flexneri was not detected in the stool of any animal for 1 year, and the clinical condition of the group was improved. Reduced recovery rates were obtained with other enteric pathogens.     

圈养猕猴志贺氏菌的分离鉴定及药敏分析

目的确定猕猴感染志贺氏菌的状况,寻找有效治疗措施。方法采用不同选择培养基对13份病猴粪便样品进行分离培养、细菌革兰氏染色、镜检,并对分离的疑似菌株进行细菌生化鉴定和分子鉴定,经小白鼠致病性实验后,再用纸片扩散法测定分离菌株对23种抗生素的敏感性。结果从13份猕猴粪便样品中共检出12株志贺氏菌,检出率为92.31%,其中痢疾志贺菌(A群)1株、福氏志贺菌(B群)10株、宋内氏志贺菌(D群)1株;致病性试验结果表明,12株菌均能在72h内致死小白鼠,并能回收到注射的菌株;药敏试验结果表明,本实验中分离到的志贺氏菌对头孢噻肟(86.49%)最敏感,对头孢三嗪(75.00%)、头孢他啶(66.67%)次之,对多粘菌素B、羧苄西林、苄唑西林素等抗生素耐药性强。结论初步确定猕猴感染志贺氏菌普遍存在,进而引起腹泻、痢疾的可能性较大,头孢噻肟等为最敏感药物。     

Partial complementation of the Fanconi anemia defect upon transfection by heterologous DNA

Summary Transfectants obtained by mouse DNA-mediated gene transfer in Fanconi anemia (FA) primary fibroblasts from the genetic complementation groups A and B were examined for the frequencies of chromosomal aberrations and cytotoxicity following treatments by cross-linking agents. Cells from group A (FA 150), which is the most sensitive to such agents, are partially corrected for both the chromosomal and cellular hypersensitivity to 8-methoxypsoralen photoaddition. In contrast, after treatment with mitomycin C (MMC), only the chromosomal sensitivity is re-established to a near normal level. The opposite is true for FA group B cells (FA 145), i.e. cell survival to MMC is partially corrected, whereas the frequency of MMC-induced chromosomal aberration remains close to that of the untransfected cells. The partial phenotypic correction of the two end points examined is interpreted as indicating either a gene dosage effect or the necessity of introducing more than one gene type in order to achieve complete recovery of a normal phenotype. The phenotypic dissociation between the clastogenic and cellular hypersensitivity to crosslinking agents may offer the opportunity of isolating separately the responsible gene(s) by conventional rescue techniques.     

Identification of Group B Streptococci by Immunofluorescence Staining

Gamma globulin fractions of rabbit antisera prepared with whole cell vaccines of group B types Ia, Ib, II, and III and labeled with fluorescein isothiocyanate stained group B streptococci type specifically. Type Ic cells, which contain the Ia polysaccharide antigen of type Ia and the Ic protein antigen of type Ib, were specifically stained by both Ia and Ib conjugates. A group B conjugate pool (B pool) that contained one conjugate specific for each group B type at its predetermined titer gave positive fluorescent-antibody (FA) reactions (4+ intensity) with group B stock strains and negative FA reactions (less than 2+ intensity) with stock strains of streptococcal groups A, C through H, and K through U, viridans streptococci, Streptococcus pneumoniae, Staphylococcus aureus, Neisseria gonorrhoeae, and representative Enterobacteriaceae. Examination of 883 clinical isolates submitted to the Streptococcus Laboratory (Center for Disease Control, Atlanta, Ga.) for identification revealed a 99.1% agreement between FA and culture-precipitin methods. All 305 group B streptococci identified by culture-precipitin and six nonhemolytic group B streptococci missed initially by culture tests were identified correctly by FA. Results of cultural and FA methods in a double-blind study of 99 vaginal swabs agreed on 96 of 99 strains. Three nonhemolytic group B streptococci were identified first by FA and later confirmed by culture-precipitin tests.     

Recovery of Bilophila wadsworthiafrom Clinical Specimens in Italy

This report is the first survey in Italy to evaluate the incidence of recovery of Bilophila wadsworthia in clinical situations. The survey was carried out at the departments of Microbiology in two Northern Italian hospitals over a one-year period. Tests for B. wadsworthia were carried out on a range of specimens from different body sites, when etiology by anaerobes was suspected. Out of a total of 350 samples examined, 67% were positive in bacteriological tests. Mixed anaerobic infections were detected in 53 specimens, corresponding to 23% of all cases. Strains of B. wadsworthia were isolated from 12 samples, equivalent to 5% and 22% of total and mixed/anaerobic infections, respectively. Bilophila wadsworthia was always isolated in mixed infections, mainly from the large intestine (67% of cases). The infectious process of B. wadsworthia was often complicated by abscess formation, regardless of body site. Interestingly, a strain was isolated from one case of bacteremia. The microorganisms most frequently isolated with B. wadsworthia were Escherichia coli for facultative species (38%), and Bacteroides fragilis, from anaerobic isolates (25%). Production of beta-lactamases by B. wadsworthia isolates was found in ten strains (83%), which appeared to be penicillin G resistant at concentration equal to or greater than the break-point (4 microg/mL). Epidemiological and clinical data from this and previous studies point to the involvement of B. wadsworthia in mixed infections. To assess the specific contribution of the species to the disease, studies of pathogenetic factors are to be considered in parallel. Nonetheless, production of beta-lactamases by most B. wadsworthia isolates could easily interfere with the therapeutical approach to infections involving the new species. The addition of a selective medium to culture specimens from the abdominal cavity should be considered in order to detect the presence of B. wadsworthia.     

Isolation of Shigellae: V. Comparison of Enrichment Broths with Stools

Many enteric media are more efficient for the detection of salmonellae than of shigellae. Comparisons of three enrichment broths and three plating media were made during analysis of 1,405 stool specimens to choose a combination of media which would enhance detection of shigellae as well. Gram-Negative (GN), Selenite, and Silliker's Broths were streaked to E M B, Salmonella-Shigella (SS), and xylose lysine deoxycholate (XLD) Agars. The enrichment broths produced a twofold increase in isolations of both salmonellae and shigellae over direct streaking. All three broths performed equally well for Salmonella detection, but GN and Silliker's produced twice as many Shigella isolates as did Selenite. Comparison of the plating media showed that XLD was markedly more efficient than either E M B or SS Agar for the recovery of both genera. SS Agar was superior to E M B for isolation of salmonellae after enrichment, whereas E M B was better for isolation of shigellae by direct streaking. Both E M B and SS were more effective when used after GN and Silliker's than after Selenite. GN Broth and XLD Agar were the most efficient combination of media. During these analyses, 158 salmonellae and 49 shigellae isolates were obtained.     

An annotated list of the mosquitoes (Diptera: Culicidae) of Mississippi

There has been no previous systematic statewide study of mosquitoes in Mississippi. This survey, resulting in the collection of over 400,000 specimens, was conducted by the authors from 2003 to 2007 throughout much of the state using CO₂‐baited CDC light traps and larval dipping. In addition, a health department contract mosquito surveillance technician collected several thousand specimens from the state from 2001 to 2003. Lastly, specimens housed at the Mississippi State University Entomological Museum, obtained from previous surveys, were included as vouchers for species occurring in the state. The collection records and literature show 60 species as occurring or having occurred in Mississippi. Voucher specimens representing 57 of the 60 species discussed are deposited in the Mississippi Entomological Museum or in the U.S. National Museum of Natural History (USNM), Washington, D.C.     

Lack of sensitivity of primary Fanconi's anemia fibroblasts to UV and ionizing radiation

Clinical observations and theoretical considerations suggest some degree of radiosensitivity in Fanconi's anemia (FA), but experimental evidence remains controversial. We tested the sensitivity of primary skin fibroblast cultures from all known FA complementation groups to ionizing radiation and ultraviolet light using conventional cell growth and colony formation assays. In contrast to previous studies, and because FA fibroblasts grow and clone poorly at ambient oxygen, we performed our sensitivity tests under hypoxic cell culture conditions. Fibroblast strains from healthy donors served as negative controls and those from patients with ataxia telangiectasia (AT) and Cockayne syndrome (CS) as positive controls. We observed interstrain variation but no systematic difference in the response of FA and non-FA control fibroblasts to ionizing radiation. After exposure to UV radiation, only complementation group A, G and D2 strains displayed values for colony formation EC50 that were intermediate between those for the negative and positive controls. Because of considerable interstrain variation, minor alterations of the response of individual FA strains to ionizing and UV radiation should be interpreted with caution and should not be taken as evidence for genotype-specific sensitivities of primary FA fibroblasts. All together, our data indicate neither systematic nor major sensitivities of primary FA fibroblast cultures of any complementation group grown under hypoxic cell culture conditions to ionizing or UV radiation.     

Comparison of Fluorescent Antibody with Cultural Technique for Isolation of Enteropathogenic Escherichia coli from Swine

In an investigation of hogs as possible reservoirs of human strains of enteropathogenic Escherichia coli (EEC), 92 six-month-old grain- and garbage-fed hogs were examined on the farm and again at the packing plant. Of the 331 specimens obtained by swabbing the rectum, cecum, and edible meat carcass of these hogs, 125 were presumptively positive for EEC when screened by the fluorescent-antibody (FA) technique. These “presumptive positive” specimens then underwent extensive bacteriological examination and complete serological typing. The FA technique proved to be an easier, simpler, and more economical procedure than culture when a large number of specimens were examined for possible EEC serogroups. It was found especially valuable for identification of multiple serogroups of EEC within a single specimen. It also appeared to be more sensitive than cultural examination, since results were not dependent on the presence of large numbers of organisms in the specimen, or even on their viability. However, the FA technique was found to be less specific than culture because of cross-reactivity with antigenically related Enterobacteriaceae when fluorescein-labeled antisera were used. Therefore, any specimen found positive on FA examination should be considered as presumptive positive until confirmed by bacteriological examination and complete serological study.     

Molecular spectra of HPRT deletion mutations in circulating T-lymphocytes in Fanconi anemia patients

The principal cellular feature of Fanconi anemia (FA), an inherited cancer prone disorder, is a high level of chromosomal breakage, amplified after treatment with crosslinking agents. Three of the eight genes involved in FA have been cloned: FANCA, FANCC and FANCG. However, their biological functions remain unknown. We previously observed an excessive production of deletions at the HPRT locus in FA lymphoblasts belonging to the relatively rare complementation group D(1) and an increased frequency of glycophorin A (GPA) variants in erythrocytes derived from FA patients (2). In thi study, we examined the molecular nature of 31 HPRT mutations formed in vivo in circulating T-lymphocytes isolated from 9 FA male patients. The results show that in all FA patients investigated the deletions are by far the most prevalent mutational event in contrast to age matched healthy donors, in which point mutations predominate. The complementation group in the FA patients examined in the present study has not yet been defined. However, knowing that mutations in the FANCA and FANCC gene are found to be involved in at least 70% of the FA patients, it can be expected that the excessive production of deletions is a general feature of the FA phenotype. In addition, the spectrum of HPRT deletions observed in FA patients differs from that of healthy children: there is a high frequency of 3'-terminal deletions and a strikingly low proportion of V(D)J mediated events. Based on previous findings, a decreased fidelity of coding V(D)J joint formation (3) and an inaccurate repair of specific DNA double strand breaks via Non-Homologous End Joining (4), we propose that FA genes play a role in the control of the fidelity of rejoining of specific DNA ends. Such a defect may explain several basic features of FA, such as chromosomal instability and deletion pronenness.     

Evaluating Burkholderia pseudomallei Bip proteins as vaccines and Bip antibodies as detection agents

Burkholderia pseudomallei is a biothreat agent and an important natural pathogen, causing melioidosis in humans and animals. A type III secretion system (TTSS-3) has been shown to be critical for virulence. Because TTSS components from other pathogens have been used successfully as diagnostic agents and as experimental vaccines, it was investigated whether this was the case for BipB, BipC and BipD, components of B. pseudomallei's TTSS-3. The sequences of BipB, BipC and BipD were found to be highly conserved among B. pseudomallei and B. mallei isolates. A collection of monoclonal antibodies (mAbs) specific for each Bip protein was obtained. Most recognized both native and denatured Bip protein. Burkholderia pseudomallei or B. mallei did not express detectable BipB or BipD under the growth conditions used. However, anti-BipD mAbs did recognize the TTSS needle structures of a Shigella strain engineered to express BipD. The authors did not find that BipB, BipC or BipD are protective antigens because vaccination of mice with any single protein did not result in protection against experimental melioidosis. Enzyme-linked immunosorbent assay (ELISA) studies showed that human melioidosis patients had antibodies to BipB and BipD. However, these ELISAs had low diagnostic accuracy in endemic regions, possibly due to previous patient exposure to B. pseudomallei.