Los Alamos Catches Clues To Dreaded Diseases

The technique helps pinpoint the source of a disease during a current outbreak, with significant implications for human health and international trade.

Los Alamos' application of the technique is reported in a paper appearing in today's (Feb. 3) issue of the Proceedings of the National Academy of Sciences. Forensic samples were analyzed from victims of a human anthrax outbreak that occurred in Russia in 1979.

Los Alamos, a U.S. Department of Energy laboratory, drew on its extensive background in the separation and analysis of genetic material to test small tissue samples from humans or animals for pathogens and identify slight variations in the genetic fragments of those pathogens.

"To distinguish different strains of a pathogen, we look at DNA fragment lengths and sequences of specific fragments," said project leader Paul Jackson. "We are looking at differences in the genome that are far less than one thousandth of one percent. And we can detect them readily."

The technique uses the polymerase chain reaction, or PCR, and DNA sequencing. PCR, which replicates DNA fragments in huge volumes, makes current genetic studies possible by providing many copies of identical fragments for study.

In a cell, the DNA polymerase enzyme plays an important role in duplicating DNA. In PCR, researchers first heat the double strands of DNA to separate the strands. Then they add the polymerase enzyme, along with compounds called "primers," which start or "prime" the reaction. The reaction builds a complementary strand of DNA for each original strand.

>From the two identical copies of double-stranded DNA, four copies are made the same way, then eight, then 16 and so on, taking advantage of exponential duplication - quickly resulting in thousands of copies for study. Thus, all the genetic material necessary for a complete analysis can be gained from one or a very few dead bacteria in a sample.

Los Alamos researchers have taken PCR to another level of precision. A double PCR strategy increased the sensitivity of the procedure while specifically selecting the pathogen under investigation. DNA products of a first PCR amplification were used as templates in a second reaction primed by primers that differentiate among strains of a species.

The technique has been tested on many pathogens. For example, there are many strains of the common bacteria Escherichia coli, but the strain E. coli 157 causes food poisoning. The Los Alamos technique can quickly identify that particular strain.

The technology is not limited to human diseases. Analysis also can identify the source of diseases in livestock, poultry and plants. Identifying a disease source or vector is critical to its control.

For example, following an outbreak of anthrax in cattle in Norway, analysis showed the Bacillus anthracis strains came from third-world countries. The list of possible source countries matched the list of countries from which the fodder supplement bone meal had been imported. Processing bone meal does not kill notoriously hardy B. anthracis spores. With a likely source of contamination identified, officials in Norway were able to reduce a serious commercial threat.

Alternately, if an analysis shows a group of samples are infected by exactly the same strain of a disease and the strain is indiginous to that location, it suggests the cause is a natural outbreak and not a food-borne infection, as was recently found in samples from Australian cattle. Such evidence can be crucial to international trade.

Los Alamos does not maintain any live B. anthracis or any other pathogenic organisms. All DNA is extracted from certified dead tissue and research is conducted on genetic fragments.

"We deal exclusively with the DNA - the genetic blueprint of an organism," Jackson said.

For the study that showed the technique works even on decades-old samples, Jackson's team performed the analysis on pencil-eraser-sized, formalin-fixed tissue samples from human anthrax victims. The study also included anthrax-infected bovine and primate samples.

The human samples came from forensic studies of an outbreak of anthrax that occurred in Sverdlovsk, Union of Soviet Socialist Republics, now Ekaterinburg, Russia, in April 1979. The event has been reported in Russian and English-language media, including a paper in Science in November 1994.

At the time, Soviet officials attributed the outbreak to consumption of contaminated meat. But evidence shows that most victims worked or lived in a narrow zone extending from a military microbiology facility in the city to the southern city limit. The zone paralleled the prevailing northerly wind.

Results from the Los Alamos analysis confirmed that tissues from all victims contained B. anthracis . Analysis showed the Sverdlovsk victims were infected by a mixture of different B. anthracis strains. Natural outbreaks of anthrax consist of one strain only. Further, they were infected by virulent B. anthracis and not a vaccine strain. A mixture of strains used as a weapon might overcome vaccines against anthrax or therapeutic agents used to combat the resulting disease.

Los Alamos National Laboratory is operated by the University of California for the U.S. Department of Energy.