Immunodot assay of Plasmodium falciparum sporozoites in mosquitoes using a direct binding membrane system.

We describe a membrane based immunodot assay for the detection of Plasmodium falciparum sporozoites mixed with mosquitoes. A crude sodium dodecyl sulfate extract of mosquitoes and sporozoites is passed through a bi-layered membrane system, the top layer being a polyvinyldiene difluoride hydrophilic pre-filter which screens out debris but allows the passage of antigen. Sporozoite, as well as mosquito, proteins are bound to the hydrophobic membrane below. This membrane was probed with a monoclonal antibody to the repeat region of the P. falciparum circumsporozoite protein, a peroxidase labeled second antibody and a tetramethyl-benzidine substrate. The method detects as few as 10 sporozoites/mosquito or 100 sporozoites in a pool of 10.

A rapid dot immunoassay for the detection of serum antibodies to eastern equine encephalomyelitis and St. Louis encephalitis viruses in sentinel chickens.

A dot enzyme-linked immunosorbent assay utilizing a novel membrane, polyvinylidene difluoride, is described. This assay was developed for the rapid detection of serum antibodies to eastern equine encephalomyelitis virus and St. Louis encephalitis virus in sentinel chickens. Antigens were spot-filtered through the membrane. Membranes were dipped into small vials of sera. Antigen-antibody complexes were detected with enzyme-conjugated antiglobulin which, when exposed to substrate, produced a colored insoluble product. The antibody detection protocol was completed within 50 min and was compared with a standard plate enzyme immunoassay. Chickens were experimentally infected with eastern equine encephalomyelitis and St. Louis encephalitis and bled on a daily basis. The dot immunoassay correctly identified 99% (123/124) of the eastern equine encephalomyelitis virus and 100% (67/67) of the St. Louis encephalitis virus antisera. Sera from sentinel chicken flocks in Maryland were also assayed. These data indicate that the dot immunoassay should be considered as an alternative to current assays for the screening of sera for antibodies to virus antigens. This assay could easily be performed in the field and allows for the screening of antibodies to several different viruses in one test.

Detection of West Nile virus by the polymerase chain reaction and analysis of nucleotide sequence variation.

A polymerase chain reaction (PCR) assay was developed to rapidly detect and identify West Nile (WN) virus. The RNA from seven isolates of WN virus from six countries and four other flaviviruses (Kunjin, Japanese encephalitis, St. Louis encephalitis, and yellow fever viruses) was reverse-transcribed (RT) and amplified by PCR. The nucleotide sequences of the amplified products were determined by a rapid, automated DNA sequencing method. The WN virus RT/PCR assay detected the target gene segment of sequencing method. The WN virus RT/PCR assay detected the target gene segment of isolates from both the African-Middle Eastern group and the Indian group with a sensitivity of approximately 0.05 pg of viral RNA. Kunjin virus was the only other flavivirus tested that produced a band of the appropriate size. Five of seven WN virus isolates showed 92-98% homology in the nucleotide sequence of their PCR products. The sequence of one isolate was virtually identical to the published sequence of the Nigerian isolate (99.5% homology). No correlation was established between the degree of nucleotide homology, geographic location, time of isolation, or source of the isolates.

Improved enzyme-linked immunosorbent assay for the detection of orbivirus antigens by treatment with sodium dodecyl sulfate.

Several methods have been developed for treating antigens, that reduce background or inactivate virus utilizing toxic reagents for the enzyme-linked immunosorbent assay (ELISA). In this study, orbivirus infected BHK-2 1 cells were treated with SDS for increased sensitivity in antigen detection by ELISA. The sensitivity of 1.0% SDS treated material increased eightfold.

A rapid membrane based immunobinding assay for the detection of dengue virus in tissue culture.

A rapid, simple dot immunoassay (DOTIA) was developed and evaluated for the detection of dengue-1 viral antigen in infected Aedes albopictus C6/36 cells. Dengue virus infected cells were solubilize in sodium dodecyl sulfate (SDS) and the lysate was pressure filtered through a hydrophobic polyvinylidene difluoride (PVDF) membrane. Viral antigen retained in the membrane was detected by a dengue-1 type specific monoclonal antibody and a peroxidase-labeled second antibody. Addition of tetramethylbenzidene (TMB) substrate produced a blue-colored precipitate which allowed for quantitation of viral antigen using a portable white light reflectance densitometer. Estimate of viral infectivity in the cell lysates tested by the DOTIA was determined by standard plaque assays and the results indicated an excellent correlation between these two methods. The dot immunoassay detected dengue viral antigen in infected C6/36 cells between days three and eight post-inoculation, depending on the titer of the inoculum. An infectivity titer of at least 10(3) plaque forming units (PFU) per ml was required to detect antigen by the DOTIA. The DOTIA also detected viral antigen in cells inoculated with twelve acute sera from known dengue-1 virus infected patients, thus demonstrating that this technique is useful for the detection and identification of dengue-1 virus from clinical specimens.

Norfloxacin compared to trimethoprim/sulfamethoxazole for the treatment of travelers' diarrhea among U.S. military personnel deployed to South America and West Africa.

A randomized treatment trial of travelers' diarrhea was carried out among U.S. military personnel participating in routine exercises in several port cities in South America and West Africa. A 5-day, twice daily course of either norfloxacin (400 mg) or trimethoprim/sulfamethoxazole (TMP/SMX, 160/800 mg) was given to 142 volunteers. At the end of 5 days of treatment, diarrhea had resolved in 100% of 73 patients receiving norfloxacin and 97.1% (67/69) receiving TMP/SMX. A probable bacterial pathogen was determined in 44% of 142 subjects: 49% of the norfloxacin group and 39% of the TMP/SMX group. The most common pathogens detected were enterotoxigenic Escherichia coli in 20% of cases and rotavirus in 15%. Resistance to TMP/SMX was present in 20 (27%) bacterial isolates, while no resistance to norfloxacin was found. Eight of 10 patients in the TMP/SMX treatment group who had TMP/SMX-resistant bacterial enteropathogens improved clinically. Both norfloxacin and TMP/SMX were clinically effective in the treatment of travelers' diarrhea in this military population.

Processing and microfiltration of mosquitoes for malaria antigen detection in a rapid dot immunobinding assay.

Data on a technique for the detection of antigen from arthropod vectors in a dot immunobinding assay are presented. In this system, antigen present in the vector was first solubilized in sodium dodecyl sulfate. The homogenate from this process was microfiltered through a two-membrane sandwich; target antigen molecules passed through the first membrane and were immobilized on the second one. The first membrane was nonbinding and served to impinge debris. The second membrane was a high-protein-binding-capacity hydrophobic polyvinylidene difluoride membrane. High signal-to-noise ratios were produced by this method, which is readily adaptable for field use. This assay was used for malaria sporozoites, but it can serve as a general technique that is applicable to other arthropod vectors and etiologic agents.

Evaluation of performance parameters of a membrane-based dot immunoassay for meningococcal polysaccharide.

Increasingly, membrane-based enzyme immunoassays are being developed as the preferred solid-phase enzyme immunoassay format. We describe the rate kinetics of a polyvinylidene difluoride membrane-based dot immunoassay for meningococcal group A polysaccharide. Antigen detection sensitivity decreased logarithmically with linear decreases in incubation time. The sensitivity of a 30-min assay (5-min incubation steps) was increased to nearly the level of the standard assay (1-h incubation steps) by increasing the concentration of assay reagents fourfold. These results support the idea that existing microtiter plate assays can be transferred to rapid dot immunoassay formats with little or no loss of sensitivity.

Tick-borne Kemerovo group orbiviruses in a Newfoundland seabird colony.

Five new isolates of Kemerovo group viruses were recovered from Ixodes uriae collected on Great Island, Witless Bay Seabird Sanctuary, Newfoundland, Canada, during July 1985. This brings the total number of Orbivirus isolates on Great Island to 18 isolates including the 7 from 1971 and 6 from 1972. Genomic segments of several strains were compared by polyacrylamide gel electrophoresis. The degree of variation in each segment of these viruses was calculated. Great Island and Bauline viruses exhibited a great degree of variation in dsRNA migration patterns. Great Island and Bauline genomes averaged 11.60 (SD = 0.107) and 11.69 megadaltons (SD = 0.075), respectively. Variation was observed in all 10 segments of Great Island and Bauline viruses. These findings were compared with serologic and protein gel data.

Identification of Campylobacter jejuni and Campylobacter coli antigens with mucosal and systemic antibodies.

The development of a rapid and specific diagnostic assay for Campylobacter infections is important in determining the etiology of acute diarrhea in humans. Studies have shown that sonicated whole bacteria or partially purified antigens cross-reacted with antibodies against other closely related bacteria. To solve the problems of specificity, we identified specific antigens of Campylobacter jejuni and Campylobacter coli for use in diagnostic assays. We investigated the responses of serum, urine, and intestinal lavage antibodies in infected (fed live bacteria) and parenterally immunized (intraperitoneal injection of sonicated whole bacteria with adjuvant) mice directed against C. jejuni or C. coli by Western blot (immunoblot) analysis. Antibody responses were examined weekly for up to 28 days. Fewer antigens were detected by urinary and intestinal lavage fluid immunoglobulin A (IgA) than serum IgG and IgM for both parenterally immunized and infected mice. Serum from parenterally immunized mice detected more antigens than that from infected mice. Two high-molecular-weight antigens (62,000 and 43,000) were predominantly detected by serum, urine, and intestinal lavage fluids of both parenterally immunized and infected mice. Serum antibodies from 28-day parenterally immunized mice detected one antigen specific to C. coli with a molecular weight of 38,000 and one antigen specific to C. jejuni with a molecular weight of 27,000. An immunodominant protein with a molecular weight of 31,000 common to both C. jejuni and C. coli was also recognized by serum antibodies from parenterally immunized mice.

Is avian adeno-associated virus an endogenous virus of chicken cells?

The adeno-associated viruses (AAV) are defective parvoviruses which produce infective progeny only in cells co-infected with a 'helper' adenovirus (Ad). Both human and simian AAV have been recovered from human and simian primary cell cultures following their inoculation with 'AAV-free' Ad. Whereas some studies have suggested that AAV exists in a latent state in these cells, others have indicated that the AAV genome is capable of establishing and maintaining a latent state in defined laboratory conditions which mimic the situation proposed for the 'latent' AAV recovered from human and simian tissues. Here, avian adeno-associated virus (AAAV) was consistently recovered from limiting dilutions of purified and unpurified avian Ad stocks propagated in embryonating chicken eggs derived from two independently raised flocks of White Leghorn (WL) chickens but not when these Ad stocks were propagated in duck cells. These observations suggest that AAAV is a latent endogenous virus of at least some flocks of WL chickens.

Retroviral transformation of cerebral microvascular endothelial cells: macrophage-like and microvascular endothelial cell properties.

We report that L-cell-conditioned medium (LCM) transforms porcine cerebral microvascular (PCMV) endothelial cells into cells with macrophage-like properties. LCM is known to contain both cytokine(s) and the L-cell virus, a murine retrovirus found in the L929 cell and LCM. Our evidence suggests that both LCM cytokine(s) and the L-cell virus are involved in this PCMV endothelial cell transformation. Criteria for transformation include focus formation, decreased serum requirements for growth, changes in morphology including nonadherence, propagation in suspension culture, and a decreased growth response to stimulation with a known endothelial cell mitogen. Macrophage-like characteristics of this transformed cell, designated as RVTE, include pinocytosis of low-density lipoprotein, Fc receptor-mediated phagocytosis, phagocytosis of bacteria and zymosan, the expression of macrophage enzyme markers, and constitutive production of colony-stimulating factor 1. However, the transformed cell retains several properties of the nontransformed cell including the expression of FVIII:RAg and in vitro self-organization into capillary-like structures. Cloning of RVTE cells clearly shows that both macrophage-like and cerebral microvascular endothelial cell properties are present in the same cell. During self-organization, nontransformed cells express morphologic and functional characteristics classically associated with the macrophage. These findings suggest that some brain capillary pathophysiologies could involve macrophage-like cerebral microvascular endothelial cells. Furthermore, the "reticuloendothelial" phenotypic repertoire expressed by this transformed cerebral microvascular endothelial cell may show that the cerebral capillary endothelial cell in vivo is derived from a hematopoietic and/or phagocytic precursor.

Detection of Francisella tularensis in blood by polymerase chain reaction.

We developed a polymerase chain reaction-based assay for Francisella tularensis which we evaluated by using spiked blood samples and experimentally infected mice. The assay detected both type A and type B F. tularensis at levels equivalent to one CFU/microliter of spiked blood. Results from polymerase chain reaction-based assay of limiting dilutions of blood from mice infected with the live vaccine strain agreed closely with results from blood culture.

Alkaline phosphatase-conjugated oligonucleotide probes for enterotoxigenic Escherichia coli in travelers to South America and West Africa.

Several studies have demonstrated the usefulness of 32P-labeled recombinant DNA probes for identifying enterotoxigenic Escherichia coli (ETEC). The use of radioisotopes and X-ray development, however, severely handicaps the utility of DNA probes in most clinical laboratories. In this study, enzyme-labeled oligonucleotide probes for ETEC LT (heat-labile toxin) and ST (heat-stable toxin) genes were compared with the standard Y1 adrenal cell and suckling mouse assays for their ability to identify ETEC in a population of American adults experiencing acute episodes of diarrhea in South America and West Africa. The LT probe hybridized with 12% (64 of 529) of the E. coli colonies tested, whereas 11% (57 of 529) were positive by Y1 adrenal cell assay. DNA from 9% (47 of 529) of the E. coli colonies tested hybridized with the ST probe, whereas only 5% (28 of 529) produced ST as measured by the suckling mouse bioassay. For the patient samples tested, correlation between probe and bioassay for LT was 97%, or three discrepancies in 111 patients tested. Overall concordance of the ST probe and bioassay was 95%, or five discrepancies in 111 patients. Enzyme-labeled oligonucleotide probes represent a major advance in the diagnosis of ETEC-associated diarrheal disease and may be used in laboratories with minimal equipment.

Detection of immunoglobulin A in urine specimens from children with Campylobacter-associated diarrhea by a chemiluminescent indicator-based western immunoblot assay.

A Western blot (immunoblot) assay was used to detect Campylobacter-specific immunoglobulin A in urine. Acute-phase urine samples from six children with Campylobacter diarrhea had titers ranging from 2 to 8. The highest titer was detected 4 days postonset. Campylobacter-specific immunoglobulin A was undetectable in the paired convalescent-phase specimens and urine samples from three control children.