Fluorescent Antibody Microscopy Research Articles

Human fecal and pathogen exposure pathways in rural Indian villages and the effect of increased latrine coverage

Efforts to eradicate open defecation and improve sanitation access are unlikely to achieve health benefits unless interventions reduce microbial exposures. This study assessed human fecal contamination and pathogen exposures in rural India, and the effect of increased sanitation coverage on contamination and exposure rates. In a cross-sectional study of 60 villages of a cluster-randomized controlled sanitation trial in Odisha, India, human and domestic animal fecal contamination was measured in community tubewells and ponds (n = 301) and via exposure pathways in homes (n = 354), using Bacteroidales microbial source tracking fecal markers validated in India. Community water sources were further tested for diarrheal pathogens (rotavirus, adenovirus and Vibrio cholerae by quantitative PCR; pathogenic Escherichia coli by multiplex PCR; Cryptosporidium and Giardia by immunomagnetic separation and direct fluorescent antibody microscopy). Exposure pathways in intervention and control villages were compared and relationships with child diarrhea examined. Human fecal markers were rarely detected in tubewells (2.4%, 95%CI: 0.3–4.5%) and ponds (5.6%, 95%CI: 0.8–10.3%), compared to homes (35.4%, 95%CI: 30.4–40.4%). In tubewells, V. cholerae was the most frequently detected pathogen (19.8%, 95%CI: 14.4–25.2%), followed by Giardia (14.8%, 95%CI: 10.0–19.7%). In ponds, Giardia was most often detected (74.5%, 95%CI: 65.7–83.3%), followed by pathogenic E. coli (48.1%, 95%CI: 34.8–61.5%) and rotavirus (44.4%, 95%CI: 34.2–54.7%). At village-level, prevalence of fecal pathogen detection in community drinking water sources was associated with elevated prevalence of child diarrhea within 6 weeks of testing (RR 2.13, 95%CI: 1.25–3.63) while within homes, higher levels of human and animal fecal marker detection were associated with increased risks of subsequent child diarrhea (P = 0.044 and 0.013, respectively). There was no evidence that the intervention, which increased functional latrine coverage and use by 27 percentage points, reduced human fecal contamination in any tested pathway, nor the prevalence of pathogens in water sources. In conclusion, the study demonstrates that (1) improved sanitation alone may be insufficient and further interventions needed in the domestic domain to reduce widespread human and animal fecal contamination observed in homes, (2) pathogens detected in tubewells indicate these sources are microbiologically unsafe for drinking and were associated with child diarrhea, (3) domestic use of ponds heavily contaminated with multiple pathogens presents an under-recognized health risk, and (4) a 27 percentage point increase in improved sanitation access at village-level did not reduce detectable human fecal and pathogen contamination in this setting.

Repeatable immersion infection with Photobacterium damselae subsp. piscicida reproducing clinical signs and moderate mortality

Pseudotuberculosis is a bacterial septicaemia caused by Photobacterium damselae subsp. piscicida in several marine fish species. Yellowtail Seriola quinqueradiata is the most sensitive fish species to this disease. The internal organs of naturally infected yellowtail exhibit whitish spots, tubercle-like tissue structures, consisting of bacterial accumulations. There have been many trials for experimental infection, however adequate method of infection that reproduces moderate mortality and primary clinical signs has not yet established. Present investigation evaluated an immersion infection method by using logarithmic culture-phase bacteria resulting in higher mortality than that using stationary culture-phase bacteria. Typical white spots on the spleen and kidney were also observed constantly in dead fish. Transmission electron microscopy and fluorescent antibody microscopy showed bacterial clusters not only in the spleen and kidney but also in the blood channels in the secondary gill filaments. These results were confirmed repeatedly by plural experiments. The use of logarithmic-phase bacteria in immersion infection is an appropriate technique to reproduce moderate mortality and primary clinical signs, which will be a reliable infection method also for the challenge test of pseudotuberculosis vaccine.

Mycobacterium smegmatishistone-like protein Hlp is nucleoid associated

Eubacteria encode proteins that are required for nucleoid organization and for regulation of DNA-dependent processes. Of these histone-like proteins (Hlps), Escherichia coli HU has been shown to associate with the nucleoid and to regulate processes such as DNA repair and recombination. In contrast, the divergent HU homologs encoded by mycobacteria have been variously identified as involved in the physiology of dormancy, in the response to cold shock, or as laminin-binding proteins associated with the cell envelope. We show here, contrary to previous reports that the HU-related Hlp from Mycobacterium smegmatis associates with the nucleoid in vivo. Using indirect fluorescent antibody microscopy we show that cold shock causes Hlp to accumulate in the cytoplasm of M. smegmatis. No evidence of surface-associated Hlp was found in M. smegmatis cells treated for cell wall permeabilization. Quantitative Western blots indicate that exponentially growing cells contain c. 120 molecules per cell, with upregulation of Hlp after cold shock estimated to be c. 10-fold. That Hlp associates with the nucleoid in vivo suggests functions in DNA metabolism, perhaps in adaptation to environmental stress.

Pneumonic plague cluster, Uganda, 2004.

The public and clinicians have long-held beliefs that pneumonic plague is highly contagious; inappropriate alarm and panic have occurred during outbreaks. We investigated communicability in a naturally occurring pneumonic plague cluster. We defined a probable pneumonic plague case as an acute-onset respiratory illness with bloody sputum during December 2004 in Kango Subcounty, Uganda. A definite case was a probable case with laboratory evidence of Yersinia pestis infection. The cluster (1 definite and 3 probable cases) consisted of 2 concurrent index patient-caregiver pairs. Direct fluorescent antibody microscopy and polymerase chain reaction testing on the only surviving patient's sputum verified plague infection. Both index patients transmitted pneumonic plague to only 1 caregiver each, despite 23 additional untreated close contacts (attack rate 8%). Person-to-person transmission was compatible with transmission by respiratory droplets, rather than aerosols, and only a few close contacts, all within droplet range, became ill.

Borrelia burgdorferi in tick cell culture modulates expression of outer surface proteins A and C in response to temperature.

The Lyme disease spirochete Borrelia burgdorferi sensu stricto downregulates outer surface protein A (OspA) and upregulates outer surface protein C (OspC) during tick feeding. The switching of these proteins correlates with increased spirochetal infectivity for the mammal. We examined the effect of temperature on differential expression of OspA and OspC by B. burgdorferi cocultivated with a cell line isolated from the vector tick Ixodes scapularis. The effect of incubation at 31, 34, or 37 degrees C on expression of OspA and OspC by B. burgdorferi JMNT and N40 was analyzed by indirect fluorescent-antibody microscopy, polyacrylamide gel electrophoresis, and immunoblotting. The amount of OspA relative to the amount of flagellin was highest in spirochetes cocultivated with tick cells at 31 degrees C and declined with increasing temperature in both strains. OspC production was enhanced in spirochetes cocultivated with tick cells at 37 degrees C. Spirochetes grown axenically in BSK-H medium also produced more OspC at 37 degrees C, but OspA content was not appreciably affected by temperature. Our findings indicate that temperature, along with cultivation in a tick cell culture system, plays a role in the differential expression of OspA and enhances differential expression of OspC by spirochetes.

Use of the Delphi Panel Method to Assess Expert Perception of the Accuracy of Screening Test Systems for Infectious Pancreatic Necrosis Virus and Infectious Hematopoietic Necrosis Virus

Fifteen people, considered to be experts on fish virology, participated in a Delphi panel exercise to solicit opinion concerning the importance of factors that influence the ability of cell culture to detect infectious pancreatic necrosis virus (IPNV) or infectious hematopoietic necrosis virus (IHNV) in asymptomatic infected salmonids. Panelists rated many factors as having a strong impact on the sensitivity of cell culture and particularly emphasized the importance of technical and laboratory-related factors. Participants also provided their perceived estimates of the sensitivity and specificity of test systems—consisting of cell culture followed by serum neutralization, specific gene probes, enzyme-linked immunosorbent assay (ELISA), or fluorescent-antibody microscopy—for IPNV and IHNV in asymptomatic salmonids. The sensitivities estimated by panelists for optimal conditions were less than 70% for both IPNV and IHNV. There was substantial panelist uncertainty about the estimates, as indicated by large variances among individual responses. The system using serum neutralization for virus identification was perceived to have the highest sensitivity. All panelists estimated specificity to be very high. The importance of these findings with respect to the design of surveillance, quality assurance and control programs, and the interpretation of screening data are discussed.

Environmental Legionella Assessment in Office Buildings Of Continental United States

An environmental assessment of Legionella bacteria was conducted in five office buildings in the continental United States where no legionellosis was reported. The purpose of this investigation was to (i) determine the presence of Legionella bacteria in potable and non-potable water systems, (ii) provide a baseline information for management, and (iii) evaluate the effectiveness of the remedial actions taken. Water samples were collected from all possible water sources in surveyed buildings. The samples were analyzed by both direct fluorescent antibody microscopy (DFA) and the bacterial cultural method for the presence of Legionella species. Legionella bacteria were detected in some samples collected from various water distribution systems in the buildings. Remedial action was taken to eliminate these bacteria, and case-by-case results are presented.

Detection of Rabies Virus Antibodies in Fruit Bats (Eidolon helvum) from Nigeria

Fruit bats (Eidolon helvum) were collected from three different localities in Western Nigeria: Oti, Ibadan and Idanre. Fifty serum samples were analyzed using a modified rapid fluorescent focus inhibition technique against rabies, Mokola, Lagos bat and Duvenhage viruses. Twenty-five brain samples were screened for rabies and related lyssavirus antigens by direct fluorescent antibody microscopy and by an indirect fluorescent antibody technique with rabies anti-nucleocapsid monoclonal antibodies. All brain samples were negative. Two serum samples had relatively high anti-rabies activity; no neutralizing activity was detected against Mokola, Lagos bat or Duvenhage viruses.

Incidence of spirochaetal infections in cases of intestinal disorder in chickens

The incidence of intestinal spirochaetes was determined using direct fluorescent antibody microscopy on faecal and mucosal samples from chickens. Of 134 flocks with intestinal disorders 27.6% were found to be positive, but only 4.4% (of 45 flocks) were positive where signs of enteritis were absent. Flocks housed in cages or with access to litter were equally affected. No evidence was found for pigs as an aetiological factor.

Experimental infection of dogs and monkeys with two rabies serogroup viruses, Lagos bat and Mokola (IbAn 27377). Gross pathologic and histopathologic changes.

Dogs and monkeys were inoculated intracerebrally or intramuscularly with Lagos bat virus or Mokola virus, two viruses serologically and morphologically related to rabies virus. All animals inoculated intracerebrally died, but some animals inoculated intramuscularly survived. One monkey inoculated intramuscularly with Lagos bat virus survived with a hemiparesis. In both dogs and monkeys that died there was a nonsuppurative meningoencephalomyelitis and ganglionitis. Lesions were more extensive than those reported after infection with street rabies virus. Intracytoplasmic inclusions, morphologically indistinguishable from Negri bodies, were seen in the central nervous system of monkeys, but not dogs, that died. With fluorescent-antibody microscopy, granular aggregations of viral antigen were observed in the cytoplasm of many neurons in the central nervous system. In some animals, the retina and ciliary body of the eye had focal inflammatory changes.

Detection of Type E Botulinal Toxin in Cultures by Fluorescent-Antibody Microscopy

Vegetative cells of toxigenic Clostridium botulinum type E cultures were stained with fluorescent antitoxin prepared against purified toxin. The staining seems to be specific.

Detection of type E botulinal toxin in cultures by fluorescent-antibody microscopy.

Vegetative cells of toxigenic Clostridium botulinum type E cultures were stained with fluorescent antitoxin prepared against purified toxin. The staining seems to be specific.

Mammary Tumor Virus (MTV) Virions in a Transplantable Ependymoblastoam

SummaryA methylcholanthrene-induced transplanted ependymoblastoma was found to be proliferating MTV virions (B particles). Identity of the particles was affirmed by thin section and negative contrast electron microscopy, by immunodiffusion precipitation against absorbed anti-MTV rabbit serum by infectivity and by fluorescent antibody microscopy of cell cultures. Cell cultures have continued to proliferate virions for a period of 10 months.

OBSERVATIONS ON ERYTHROCYTIC INCLUSION BODIES FROM MICE INFECTED WITH RABIES VIRUS.

SummaryRabies was induced in Webster-Swiss mice injected intramuscularly with 0.1 ml of a mouse brain suspension of rabies virus, the titer of which was 10-1.75. Peripheral blood films stained by Shorr's modified technique and Kleineberger-Nobel's method showed approximately 1.0% of the erythro-cytes to contain discrete intracytoplasmic inclusions. The highest incidence of these bodies appeared at postinoculation day 8, with most deaths occurring at postinoculation day 13. The specificity of these inclusions has not been established. While these inclusions are related to the development of rabies in the host, it has not been demonstrated conclusively by either fluorescent antibody microscopy or mouse inoculation that these bodies are due to infection with rabies virus per se.