376A Volume 54, Number 11, 2000
focal pointBY LAURA A. VANDERBERG
LOS ALAMOS NATIONAL LABORATORYLOS ALAMOS, NEW MEXICO 87545
Detection of BiologicalAgents: Looking forBugs in All theWrong Places
T he threat of new and potentpathogens has become a greatconcern over the last severalyears. Recent advances in biotech-nology, the low cost and ease of pro-ducing potent pathogens, and theirrelative invisibility have increasedthe likelihood for biowarfare. Al-most any pathogenic organism canbe used as a biological warfare (BW)agent. Table I shows some of themore common pathogenic microor-ganisms that may be employed asagents of biowarfare. Currently, atleast seven countries are thought tohave active biothreat agent (BA)production and research programs.1
On a weight-for-weight basis, BAsare more toxic than chemical warfare(CW) agents and can potentially pro-vide broader coverage than CWagents per pound of payload. Smallquantities of biological material (forexample, 1 kg of anthrax) may harmhundreds of thousands of people, de-pending on the delivery method andweather conditions.2
The cost to produce BAs is mini-
mal and does not require any specialequipment. In fact, the same equip-ment that is used to produce biotech-nological products (e.g., fermenta-tion systems, centrifuges, chroma-tography column puri cation equip-ment, autoclaves, cell concentrators)can be used to produce BAs! Thisdual-use issue is daunting in terms ofbeing able to detect proliferation ofbiological weapons research. Oncedisseminated, BAs can reproduce inthe host to cause infection and befurther disseminated.
History. One of the rst recordeduses of biological warfare was in1346, when bodies of Tartar soldierswho had died of the plague were cat-apulted over the walls of Kaffa, a be-sieged city. During the French andIndian War (17541767) smallpoxwas used against Native Americans.A captain in the British forces gaveblankets and a handkerchief from thesmallpox hospital to the NativeAmericans and recorded an entry inhis diary that read, I hope it willhave the desired effect.3 Both ofthese attempts to wage biologicalwarfare happened at about the same
time as outbreaks (epidemics) of thesame diseases, respectively. Thus,the plague outbreak in Kaffa and thesmallpox epidemic among NativeAmericans could conceivably havebeen the result of natural outbreaksand not due solely to these inci-dents.4
World War I enemies took advan-tage of microbiological advancesand employed biological warfare atwill. The Germans supposedly in-fected livestock that were to be ex-ported to Allied forces with B. an-thracis and B. mallei, and the U.S.attempted to contaminate livestockfeed.5,6 Despite the international dip-lomatic effort to limit proliferationof weapons of mass destruction(both chemical and biological) fol-lowing the war, a number of coun-tries began research efforts to devel-op biological weapons. These in-cluded Poland, the Netherlands, Bel-gium, France, the Soviet Union, andItaly.7
The Japanese used biologicalweapons against the Manchuriansduring 19321945. Examples oftheir use included contaminating wa-
APPLIED SPECTROSCOPY 377A
FIG. 1. Diagram of a (A) prokaryotic and (B) eukaryotic cell with major componentslabeled. Note that the prokaryotic (bacterial) cell is essentially a baggie of mole-cules, whereas the eukaryotic (mammalian, plant, etc.) cell is organized into discreetorganelles.
ter and food supplies in at least 11Chinese cities with a number of dif-ferent pathogenic bacteria and re-leasing plague-infested eas overChina.4
The United States began an offen-sive bio logical weapons researchprogram in 1942 and by the late1960s had amassed a formidable ar-senal of weapons including agentssuch as B. anthracis, botulinum tox-in, F. tularensis, and several anti-
crop agents. Despite the allegationsof deployment against several coun-tries , these weapons were neverused. The U.S. program was termi-nated in 1969, and stocks of the bi-ological weapons were destroyedduring 19711973.4
The rati cation of the BiologicalWeapons Convention (BWC) in1972 has not brought an end to theproliferation of biological warfare.In fact, several countries that signed
the BWC have apparently continuedtheir programs. In one notable inci-dent, the Soviets allegedly used ri-cin, a potent toxin produced in castorbeans, to execute a Russian defector.Ricin was placed into a drilled-outpellet, sealed with dissolvable wax,and red from a weapon disguised asan umbrella.8
Detection. Detection of a BA at-tack is extremely dif cult. Moreover,effective detection for warning of abiological attack must be both ex-tremely fast and very sensitive as thepresence of as few as 10 organismsmight be an infectious dose.9 Speci- city is critical since attacks are like-ly to occur in complex environmen-tal backgrounds, some of which con-tain naturally occurring pathogens orclose relatives to the pathogen of in-terest.
The ease with which these agentscan be produced by using traditionalbiotechnology industry equipment orsimple home-brew ing equipmentmakes detection quite dif cult. Inaddition, the human senses have nomeans to recognize when exposurehas occurred, and the delay in onsetof symptoms makes identi cation ofthe place and time of attack dif cult.BW attacks may resemble and be at-tributed to a natural outbreak of adisease, particularly if a country isnot at war. In addition, the environ-mental background against whichbiothreat agents must be detected isbiologically complex, and manynonbiological particles may interferewith various detection schemes. Thebackground might also contain nat-urally occurring populations of theBA that one is attempting to detect.
The aim of this Focal Point articleis to explain the biological basis forBA detection and provide some ex-amples of spectroscopic methods forsuch. In particular, detection of theentire bacterial cell and excreted bio-molecules is presented.
A Little Microbiology. Bacteriaare fairly simple single-celled organ-isms. Bacterial cells are generallycategorized into three shapesrods,cocci, or spirellaand they are
378A Volume 54, Number 11, 2000
FIG. 2. IR spectral contours from a pathogen, Morganella morganii. Spectral range1200900 cm21. (A) Original spectrum normalized to equal absorbance; (B) rst deriv-ative of A; (C) second derivative of A. Reprinted with permission from Naumann et al.,1988.
TABLE I. BA bacteria, rickettsia, and fungi.
Lethality ifuntreated (from
BacteriaBacillus anthracisClostridium botulinumYersinia pestisBrucella melitensisFracisella tularensisVibrio choleraCorynebacteriumdiphtheriae
Burkholderia malleiSalmonella typhi
RickettsiaCoxiella burnetiiRickettsia prowazekiRickettsia mooseriRickettsia rickettsi
Q feverEpidemic typhusEndemic typhusRocky mountainspotted fever
FungiCoccidioides immitisTilletiaPuccinia graminis
CoccidiodomycosisWheat smutWheat smut
a Crop pathogens.
structurally different from mamma-lian cells (Fig. 1). Shape is main-tained by a cell wall, and the selec-tively permeable boundary betweenthe cell and the environment is itscell membrane. Other structuralcomponents (i.e ., enzymes, ribo-somes, and nuclear material) arefound in the cytoplasm, the aqueous uid of the cell where metabolismtakes place.
The cell wall is a rigid structurepredominantly made of peptidogly-can, a polymer composed of N-ace-tylglucosamine and N-acetylmura-mine cross linked by short peptides.Bacillus anthracis, the causativeagent of anthrax, is a Gram positivemicrobe and contains a thick layer ofpeptidoglycan. Yersinia pestis, thecausative agent of the plague, is aGram negative microbe and has athin layer of peptidoglycan. Coxiellaburnetti, the causative agent of Q fe-ver, is a rickettsial organism, some-thing like a cross between bacteriaand viruses. The organisms have acell membrane, but are completelydependent upon their host for surviv-al.
Some bacteria can form dormantstructures, spores, that are formedunder adverse environmental condi-tions. These are highly dessicatedstructures, akin to a nut, with severallayers of protection. The core con-tains the cell proper, dipicolinic acid(DPA), a chemical unique to bacte-rial spores, and calcium ions. Thedipicolinic acid and calcium ions arethought to provide heat resistance.The next layer is the cortex, and sur-rounding it is the spore coat, com-posed of densely packed, less cross-linked peptidoglycan.10 Spores aremetabolically inactive and have tre-mendous heat, chemical, and radia-tion resistance. When conditions be-come favorable (i.e., when there isfood availab le), spores germinateand become vegetative cells onceagain.
The cell membrane, found in allmicrobes, is a uid structure com-posed of phospholipids and proteins.As many as seven different phospho-lipids and 200 proteins have beenfound in the membrane of common
APPLIED SPECTROSCOPY 379A
bacteria such as Escherichia coli.11
The interactions of these differentproteins and phospholipids may pro-vide a diagnostic ngerprint for aparticular microbe.12
Many intracellular molecules inbiological systems (not just bacteria)are associated with energy-yieldingreactions. Some have speci c elec-tronic excitation and emission spec-tra, providing a spectroscopic signa-ture. For example, the amino acidstryptophan, phenylalanine, tyrosine,and histidine, which are componentsof proteins, can be excited by radi-ation at 250300 nm. Nicotinamideadenine dinucleo tide (phosphate)