**Staphylococcal Enterotoxins, Stayphylococcal Enterotoxin B and Bioterrorism**

Martha L. Hale

*United States Army Research Institute of Infectious Diseases, Integrative Toxicology Division, Fort Detrick, USA* 

#### **1. Introduction**

40 Bioterrorism

Tang, S., Moayeri, M., Chen, Z., Harma, H., Zhao, J., Hu, H.,Purcell, R.H., Leppla, S.H., and

Thayer, A., (2003), Homeland Security: Postal Service Readies Defense - Team will install PCR-based systems to detect biohazards in mail facilities, *C&EN*, 81, p. 7. Weaver, M.J., Farquharson, S., & Tadayyoni, M.A., (1985), Surface-enhancement factors for

Woodruff, W.H., Spiro, T.G., & Gilvarg, C., (1974), Raman Spectroscopy In Vivo: Evidence

Using Europium Nanoparticles, Clin.Vacc. Immun., 16, p. 408.

electrode structure, *J. Chem. Phys.*, 82, p. 4867.

*Biophys. Res. Commun.*, 58, p. 197.

Hewlett, I.K.,(2009) Detection of Anthrax Toxin by an Ultrasensitive Immunoassay

Raman scattering at silver electrodes: Role of adsorbate-surface interactions and

on the Structure of Dipicolinate in Intact Spores of *Bacillus Megaterium, Biochem.* 

Staphylococcal enterotoxins (SEs) are exotoxins produced primarily by *Staphylococcus aureus,* which is a ubiquitous microorganism with world-wide distribution (Bergdoll, 1983; Dinges et al., 2000). SEs are a major cause of food poisoning and they are also potent immune activators that lead to serious immune dysfunction (Alouf and Muller-Alouf, 2003; McCormick et al., 2001). Unlike most toxins, SEs are not directly cytotoxic and cell entry is not a requirement for them to cause an effect. The Centers for Disease Control and Prevention (CDC) place one SE, staphylococcal enterotoxin B (SEB), as a select agent based on its universal availability, ease of production and dissemination, and the potential to cause moderate but widespread illnesses. Additionally, because these agents are common to the environment and the diseases they cause are similar to other diseases, Category B agents require close environmental monitoring and enhanced disease surveillance (http://www.bt.cdc.gov/bioterrorism/).

Many biothreat agents are common inhabitants of the soil and animals, and are known to cause disease in areas where they are indigenous. SEs present an additional problem in that SE-producing *S. aureus* are found throughout the world, and are known to produce a variety of illnesses, so that detection of a possible bioterrorist attack may be more problematic than those of other agents (Ahanotou, et al., 2006). The following sections describe SEB's history as a biowarfare agent and its possible use as a bioterrorism agent. To understand why it is considered a Category B agent, a description of the toxin, the main diseases caused, methods to treat the diseases, and surveillance mechanisms will also be discussed.

#### **2. Biowarfare history**

In the era of offensive biological weapons, one of the SEs, SEB, was studied, not so much for its mass destruction capabilities but, rather for its ability to incapacitate soldiers so they would be incapable of fighting or defending their posts (Croddy and Hart, 2002; Hursh et al., 1995). The United States bioweapons program studied the toxin intensively and determined that the amount of SEB required to induce incapacitation was considerably less than that of synthesized chemicals. When the toxin and chemicals were compared by expense, time, and complexity of production, SEB was far more cost-effective. A dose of 400 pg/kg body weights was estimated to incapacitate 50% of the human population exposed

Staphylococcal Enterotoxins, Stayphylococcal Enterotoxin B and Bioterrorism 43

Fig. 1. Molecular structure of SEB (from PDB:3SEB using Jmol version 12.0.41) showing αhelices (magenta), β-strands (gold), and loop strucuture. The β-grasp domain is on the left,

from *S. aureus* (Table 1). Using the Clustal W program, the amino acid sequences of the SEs were aligned and evolutionary distances determined. A dendrogram constructed by the near neighbor-joining method divides the toxins into three major and two minor monophyletic groups (Ono et al., 2008; Uchiyama et al., 2003). The first two groups contain the classical toxins SEA, SED, SEE (Group 1) and SEB, SEC (Group 2) in addition to newly identified SEs; Group 3 contains only newly identified toxins. There is some similarity in structure in that

Many, but not all, SEs require zinc ions for functional binding to the MHC class II and for stability of its tertiary structure (Fraser et al., 1992; Ples et al., 2005); related to their amino acid sequences, the SEs bind zinc at various locations within the molecule (Brosnahan et al., 2010). Some bind zinc in the concave β sheet of the C terminal domain while others bind zinc in a cleft between the two domains. SEB and toxic shock syndrome toxin (TSST-1 do not bind zinc ions (Brosnahan and Shlievert, 2011; Ly, et al., 2001; Sundstrom, et al, 1996).

Analysis of SE genes indicates divergence from a common ancestry. Most genes coding for the enterotoxins are found on mobile elements such as pathogenicity islands, plasmids and bacteriophages, making horizontal transfer a common occurrence (Jarraud et al., 2001; McCormick et al, 2001; Yarwood, et al., 2002). In 2001, a cluster of genes with homologies to SE genes was identified and named the enterotoxin gene cluster (egc). Since many of the genes produced SE-like proteins, Jarraud et al. (2001) suggested that the gene cluster formed an enterotoxin nursery where genomic rearrangements would lead to new SEs, a fact that has now been confirmed with the development of the new exotoxin SEG (Lindsay, 2011;

and the disulfide bond in the right. The N terminus and C terminus are labeled.

Groups 1, 2, and 5 have a disulfide bond while Group 3 and Group 4 (TSST-1) do not.

**3.2 Genetic analysis of SE genes** 

Thomas et al., 2006).

by an aerosol attack, while 200 ng/kg body weights would be lethal for 50% of those exposed (Ahanotu, et al., 2006; Bellamy and Freedman, 2001; Ulrich et al., 1997).

By 1966, the U.S. and its allies had produced stockpiles of various biowarfare (BW) agents, including SEB (under the code name WG) and research to establish parameters for SEB's use as an aerosolized bioweapon continued at several facilities in the U.S. and Great Britain. In the fall of 1969, President Nixon stopped the offensive BW program and by 1972, all stockpiles of agents were destroyed (Greenfield et al., 2002; Franz et al., 1997). On April 10, 1972, Great Britain, United States, and Soviet governments signed the Convention on the Prohibition of the Development, Production and Stockpiling of Bacteriological (Biological) and Toxin Weapons and on Their Destruction, which went into effect March 26, 1975.

During the latter half of the Cold War, the Defense Intelligence Agency (DIA) and the Central Intelligence Agency (CIA) suspected that the USSR was continuing to stockpile and test biological weapons and therefore, defensive research programs were established for vaccine and therapeutic development (Ulrich et al., 1997). Not only have these research programs aided in development of surveillance mechanisms, the programs have significantly contributed to a greater understanding of diseases and the development of possible therapeutic interventions.

With the end of the Cold War and dissolution of the USSR, threat of BW was greatly diminished. Other rogue nations were still stockpiling weapons and the CIA uncovered evidence that Iraq was building an arsenal of biological weapons. Although weaponized SEB was considered a high probability, it was not found when the Iraqi weapons program was dismantled (Zalinskas, 1997).

#### **3. The toxin**

SEB is one of several exotoxins isolated from *S. aureus* that are known for their emetic and superantigen traits (Bergdoll et al., 1974; McCormick et al. 2001). These exotoxins were the first superantigens to be identified, but since their discovery, additional superantigens have been isolated in other bacteria, particularly from the closely related genus, *Streptococcus*. Although staphylococcal and streptococcal superantigens are very similar, descriptions of toxin here will be limited to those toxins produced by staphylococci and the toxins will be identified as SEs or superantigens (SAG).

#### **3.1 Description of the toxin**

SEB belongs to a group of pyrogenic enterotoxins, produced primarily by *S. aureus* (McCormick et al., 2001). They are water soluble and relatively resistant to heat and proteolytic enzymes, including pepsin, trypsin, and papain (Le Loir et al., 2003). Stability does also depend upon purity of the toxin preparation, the medium's composition, and the pH. SEB is one of the most stable toxins when exposed to extreme temperature and pH, one characteristic that makes SEB an attractive bioterrorism agent (da Cunha et al., 2007; Le Loir et al., 2003; Nout et al., 1988).

Although they vary in amino acid sequence, SEs share a common three-dimensional structure that maintains their unique binding regions (Fig. 1) (Baker and Acharya, 2004; Papageorgiou et al., 1998). At least 20 serologically distinct SEs have been isolated primarily

by an aerosol attack, while 200 ng/kg body weights would be lethal for 50% of those

By 1966, the U.S. and its allies had produced stockpiles of various biowarfare (BW) agents, including SEB (under the code name WG) and research to establish parameters for SEB's use as an aerosolized bioweapon continued at several facilities in the U.S. and Great Britain. In the fall of 1969, President Nixon stopped the offensive BW program and by 1972, all stockpiles of agents were destroyed (Greenfield et al., 2002; Franz et al., 1997). On April 10, 1972, Great Britain, United States, and Soviet governments signed the Convention on the Prohibition of the Development, Production and Stockpiling of Bacteriological (Biological) and Toxin Weapons and on Their Destruction, which went into effect March 26, 1975.

During the latter half of the Cold War, the Defense Intelligence Agency (DIA) and the Central Intelligence Agency (CIA) suspected that the USSR was continuing to stockpile and test biological weapons and therefore, defensive research programs were established for vaccine and therapeutic development (Ulrich et al., 1997). Not only have these research programs aided in development of surveillance mechanisms, the programs have significantly contributed to a greater understanding of diseases and the development of

With the end of the Cold War and dissolution of the USSR, threat of BW was greatly diminished. Other rogue nations were still stockpiling weapons and the CIA uncovered evidence that Iraq was building an arsenal of biological weapons. Although weaponized SEB was considered a high probability, it was not found when the Iraqi weapons program

SEB is one of several exotoxins isolated from *S. aureus* that are known for their emetic and superantigen traits (Bergdoll et al., 1974; McCormick et al. 2001). These exotoxins were the first superantigens to be identified, but since their discovery, additional superantigens have been isolated in other bacteria, particularly from the closely related genus, *Streptococcus*. Although staphylococcal and streptococcal superantigens are very similar, descriptions of toxin here will be limited to those toxins produced by staphylococci and the toxins will be

SEB belongs to a group of pyrogenic enterotoxins, produced primarily by *S. aureus* (McCormick et al., 2001). They are water soluble and relatively resistant to heat and proteolytic enzymes, including pepsin, trypsin, and papain (Le Loir et al., 2003). Stability does also depend upon purity of the toxin preparation, the medium's composition, and the pH. SEB is one of the most stable toxins when exposed to extreme temperature and pH, one characteristic that makes SEB an attractive bioterrorism agent (da Cunha et al., 2007; Le Loir

Although they vary in amino acid sequence, SEs share a common three-dimensional structure that maintains their unique binding regions (Fig. 1) (Baker and Acharya, 2004; Papageorgiou et al., 1998). At least 20 serologically distinct SEs have been isolated primarily

exposed (Ahanotu, et al., 2006; Bellamy and Freedman, 2001; Ulrich et al., 1997).

possible therapeutic interventions.

was dismantled (Zalinskas, 1997).

identified as SEs or superantigens (SAG).

**3.1 Description of the toxin** 

et al., 2003; Nout et al., 1988).

**3. The toxin** 

Fig. 1. Molecular structure of SEB (from PDB:3SEB using Jmol version 12.0.41) showing αhelices (magenta), β-strands (gold), and loop strucuture. The β-grasp domain is on the left, and the disulfide bond in the right. The N terminus and C terminus are labeled.

from *S. aureus* (Table 1). Using the Clustal W program, the amino acid sequences of the SEs were aligned and evolutionary distances determined. A dendrogram constructed by the near neighbor-joining method divides the toxins into three major and two minor monophyletic groups (Ono et al., 2008; Uchiyama et al., 2003). The first two groups contain the classical toxins SEA, SED, SEE (Group 1) and SEB, SEC (Group 2) in addition to newly identified SEs; Group 3 contains only newly identified toxins. There is some similarity in structure in that Groups 1, 2, and 5 have a disulfide bond while Group 3 and Group 4 (TSST-1) do not.

Many, but not all, SEs require zinc ions for functional binding to the MHC class II and for stability of its tertiary structure (Fraser et al., 1992; Ples et al., 2005); related to their amino acid sequences, the SEs bind zinc at various locations within the molecule (Brosnahan et al., 2010). Some bind zinc in the concave β sheet of the C terminal domain while others bind zinc in a cleft between the two domains. SEB and toxic shock syndrome toxin (TSST-1 do not bind zinc ions (Brosnahan and Shlievert, 2011; Ly, et al., 2001; Sundstrom, et al, 1996).

#### **3.2 Genetic analysis of SE genes**

Analysis of SE genes indicates divergence from a common ancestry. Most genes coding for the enterotoxins are found on mobile elements such as pathogenicity islands, plasmids and bacteriophages, making horizontal transfer a common occurrence (Jarraud et al., 2001; McCormick et al, 2001; Yarwood, et al., 2002). In 2001, a cluster of genes with homologies to SE genes was identified and named the enterotoxin gene cluster (egc). Since many of the genes produced SE-like proteins, Jarraud et al. (2001) suggested that the gene cluster formed an enterotoxin nursery where genomic rearrangements would lead to new SEs, a fact that has now been confirmed with the development of the new exotoxin SEG (Lindsay, 2011; Thomas et al., 2006).

Staphylococcal Enterotoxins, Stayphylococcal Enterotoxin B and Bioterrorism 45

contribute to the number and genetic variation within this group of toxins. Interestingly, however, diversity in amino acid sequences has not affected toxin binding to its receptors, suggesting that as the proteins evolved, selective pressures maintained their binding sites by keeping a tertiary structure that supports the characteristic binding (Baker and Achara, 2004;

Expression of SE genes is highly regulated by growth phase and environmental conditions, and not all conditions are suitable for gene activation (Lindsay, 2011). With the proper medium, most toxin production occurs in late log or stationary phase (Otero et al., 1990; Rahkovic et al., 2006; Soejima et al, 2007). *S. aureus* produces regulatory proteins and small RNAs that control toxin production, probably so that in harsh conditions, the bacterium can

One major regulator of some, but not all SE toxin production, is the accessory regulator gene (Agr) system (Lindsay, 2011). When bacteria reach a critical mass, a quorum-sensing system activates Agr, which, in turn, activates some toxin genes (SEB, for example). Other regulatory systems such as SarA can also up-regulate toxin genes indicating that regulatory

Marrack and Kappler (1990) coined the term "superantigen" to connote the similarities between SEs and conventional protein antigens that activate T cells by cross-linking T cells to antigen-presenting cells (APC). Both superantigens (SAG) and conventional antigens bind to the major histocompatibility class II (MHC class II) receptor located on APCs (Haffner, et al., 1996). However, conventional antigens bind to MHC class II molecules inside their antigen-binding grove and are processed into peptides expressed on the cell surface before they are presented to T cells via the T-cell receptor (TCR). In contrast, SAGs bind directly to MHC class II molecules outside the antigen-binding groove; they are not processed into

Fig. 2. Conventional antigen and SAGs bind APC and TCR. (A) SAGS bind to the outer region of the TCR Vβ and to the outer region of the MHC Class II determinant. (B) Conventional antigens are processed into peptides that are then presented to the antigen-

pathways are complex and multiple systems probably control toxin production.

Ulrich et al., 2007).

conserve energy (Horsburg, 2008; Fournier, 2008).

**3.3 Superantigen characteristics of SEB** 

binding region of the TCR.

peptide fragments before presentation to TCRs (Fig. 2).


aStaphilococcal enterotoxins are divided into 5 monophyletic groups according to ammino acid sequence alignment (Uchiyama et al., 2003; Ono et al., 2008)

bGene location of the toxin

centerotoxin gene cluster

dStaphylococcus aureus phatogeniciti islands

Table 1. Staphylococcal enterotoxin/superantigens

As shown in Table 1, genetic elements containing SE genes vary. Most are located on mobile genetic elements (MGEs) which are DNA pieces with ends that encode genes (Lindsay, 2011). There are several types of MGEs, including plasmids, pathogenicity islands, bacteriophages, and transposons. MGEs move from one bacterium to another or between various genetic elements in the same bacterium. Mobility of the genes is thought to contribute to the number and genetic variation within this group of toxins. Interestingly, however, diversity in amino acid sequences has not affected toxin binding to its receptors, suggesting that as the proteins evolved, selective pressures maintained their binding sites by keeping a tertiary structure that supports the characteristic binding (Baker and Achara, 2004; Ulrich et al., 2007).

Expression of SE genes is highly regulated by growth phase and environmental conditions, and not all conditions are suitable for gene activation (Lindsay, 2011). With the proper medium, most toxin production occurs in late log or stationary phase (Otero et al., 1990; Rahkovic et al., 2006; Soejima et al, 2007). *S. aureus* produces regulatory proteins and small RNAs that control toxin production, probably so that in harsh conditions, the bacterium can conserve energy (Horsburg, 2008; Fournier, 2008).

One major regulator of some, but not all SE toxin production, is the accessory regulator gene (Agr) system (Lindsay, 2011). When bacteria reach a critical mass, a quorum-sensing system activates Agr, which, in turn, activates some toxin genes (SEB, for example). Other regulatory systems such as SarA can also up-regulate toxin genes indicating that regulatory pathways are complex and multiple systems probably control toxin production.

#### **3.3 Superantigen characteristics of SEB**

44 Bioterrorism

SEB 28.4 SaPId

1

**Groupa Enerotoxin**

2

a

bGene location of the toxin centerotoxin gene cluster

3 SEI 24.9 egc poor

4 TSST-1 21.9 PI no 5 SET 22.6 egc poor

As shown in Table 1, genetic elements containing SE genes vary. Most are located on mobile genetic elements (MGEs) which are DNA pieces with ends that encode genes (Lindsay, 2011). There are several types of MGEs, including plasmids, pathogenicity islands, bacteriophages, and transposons. MGEs move from one bacterium to another or between various genetic elements in the same bacterium. Mobility of the genes is thought to

aStaphilococcal enterotoxins are divided into 5 monophyletic groups according to ammino acid

sequence alignment (Uchiyama et al., 2003; Ono et al., 2008)

Table 1. Staphylococcal enterotoxin/superantigens

dStaphylococcus aureus phatogeniciti islands

SEA 27.1 phage yes SED 26.9 plasmid (pIB485) yes SEE 29.6 prophage yes SEJ 31.2 plasmid (pIB485) yes SEN 26.1 egcc yes SEO 26.8 egc yes SES 26.2 phage yes SEP 26 phage yes SHEc 25.1 transposon yes

**MW (kDa) Gene Locationb Emetic** 

SEC1 27.5 SaPI yes SEC2 27.6 SaPI yes SEC3 27.6 SaPI yes SEG 27 egc yes SEIR 27 plasmid (pIB485) yes SEU 27.2 egc yes

SEK 26 SaPI yes SEL 26.8 SaPI no SEM 24.8 egc yes SEQ 26 SaPI unknown

yes

Marrack and Kappler (1990) coined the term "superantigen" to connote the similarities between SEs and conventional protein antigens that activate T cells by cross-linking T cells to antigen-presenting cells (APC). Both superantigens (SAG) and conventional antigens bind to the major histocompatibility class II (MHC class II) receptor located on APCs (Haffner, et al., 1996). However, conventional antigens bind to MHC class II molecules inside their antigen-binding grove and are processed into peptides expressed on the cell surface before they are presented to T cells via the T-cell receptor (TCR). In contrast, SAGs bind directly to MHC class II molecules outside the antigen-binding groove; they are not processed into peptide fragments before presentation to TCRs (Fig. 2).

Fig. 2. Conventional antigen and SAGs bind APC and TCR. (A) SAGS bind to the outer region of the TCR Vβ and to the outer region of the MHC Class II determinant. (B) Conventional antigens are processed into peptides that are then presented to the antigenbinding region of the TCR.

Staphylococcal Enterotoxins, Stayphylococcal Enterotoxin B and Bioterrorism 47

1960; Bergdoll et al., 1959; Bergdoll et al., 1965; Casman et al., 1967). Since the characterization of the five serotypes, at least 20 more SEs (Table 1) have been isolated and characterized with many inducing emesis in monkeys or humans (Uchiyama et al., 2003).

**Enerotoxin TCR Vβ Specificity** 

SEC1 3,6.4,6.9,12,13.2,14,15,17,20

SEA 1.1,5.3,6.3,6.4,6.9, 7.3,7.4,9.1,18 SEB 3,12,14,15,17,20

SEC2 12,13.2,14,15,17,20 SEC3 3,5,12,13.1,13.2

SEE 5.1,6.3,6.4,6,9,8.1,18

SED 5,12

SEJ ND

SEG 13.6,14,15 SHE Vα10

SEK 5.1,5.2,6.7 SEL 5.1,5.2,6.7,16,22

SEM 18,21.3

SEO 5.1,7,22

SEU 13.2,14

TSST-1 2,4

SEIP 5.1,6,8,16,18,21.3 SEQ 2,5.1,5.2,6.7,21.3 SEIR 3,11,12,13.2,14

Table 2. Staphylococcal enterotoxins showing the corresponding TCR V repertoires

Approximately 25% of healthy people and animals carry *S. aureus* on the skin and often food workers who carry the bacterium may contaminate food when they handle food without washing their hands or wearing gloves. The microorganism is also found in unpasteurized milk and cheese products and, being salt tolerant, grows in salty foods as well. Because they are highly resistant to heat and enzymatic inactivation, foods that do not require cooking or those prepared by hand provide greater risks of contamination with the bacteria and subsequent toxin production. The short incubation period, approximately 4-6 hr after ingestion, usually differentiates SE-induced food poisoning from those caused by bacteria such as *E. coli* or *Salmonella* species where presence of the bacteria is required for disease.

SEN 9

SEI 1,5.1,5.2,5.3,23

While SAG must first bind to MHC class II molecules, their ability to activate large populations of T cells depends largely upon their binding to the TCR (Alber et al., 1990; Pontzer, et al., 199; Stevens, 1997; White et al., 1989). There are five different elements (Vα, Jα, Vβ, Dβ, and Jβ) that comprise the two-chain TCR receptor with each composed of a constant and variable region. The TCR uses all five elements for recognition of processed peptides on the surface of APCs (from conventional antigens). Peptide binding occurs within the peptide-binding region and requires a helper molecule such as CD4. Superantigens bind only to Vβ elements and specificity of binding results from the types of Vβ molecules present (Fig. 1). For example, SEB binds to human Vβ phenotypes 3, 12, 14, 15, 17, which differ from phenotypes bound by other SAGs (Table 2). Because many T cells contain the same Vβ phenotype, SAGs may activate up to 20% of the whole T-cell population rather than the 0.01% activation by conventional antigens.

SAGs are not restricted and can bind to both CD4 and CD8 T cells if the SAG recognizes the TCR Vβ chains, which increases the number of T cells they can affect. Superantigenactivated T cells can undergo at least five to six rounds of cell division (Nagshima et al., 2004). The massive clonal T-cell expansion results in the activation of programmed cell death in which cells responding to the specific superantigen are deleted (Choi et al., 1989; Yuh et al., 1993). Apoptosis of large numbers of T cells, clonal deletion and anergy of specific T-cell populations, and massive release of proinflammatory cytokines are all factors in the toxin's pathogenesis.

#### **4. SE pathogenesis**

There are two major diseases caused by the staphylococcal enterotoxin superantigens (SEs), and most diseases attributed to the SEs relate to chronic disease states caused by autoimmunity and repetitious stimulation by SEs. Those autoimmune diseases in which SEs are thought to play a role require more than one challenge and therefore, will probably not be a concern in a bioterrorism threat. The two diseases that are pertinent to potential bioterrorism attacks are SEB (or SEs) in the food or water supply (food poisoning) or an aerosol attack in which the toxin will be inhaled into the lungs, possibly causing toxic shock syndrome.

#### **4.1 Food poisoning**

As noted previously, *S. aureus* is a ubiquitous microorganism with a world-wide distribution, and is responsible for causing large numbers of food poisoning cases throughout the world (Bergdoll, 1989; Le Loir et al., 2003; Ortega, et al., 2010). In its normal environment, the gram-positive cocci and its toxins do not cause disease; however, when introduced into foods such as cream, mayonnaise, or similar foods, the bacteria grow rapidly secreting the exotoxins which then contaminate the food. Dack and coworkers (1930) provided the first documented report that identified a toxin from *S. aureus* as a causative agent of a food poisoning incident involving staphylococci-contaminated Christmas cake. The investigators grew the bacteria isolated from the cake and found that a sterile filtrate from the broth in which the bacteria were grown induced food poisoning when ingested by human volunteers. Thereafter, from investigations of various food poisoning outbreaks, an initial five antigenically distinct enterotoxins were identified suggesting that *S aureus* produced a family of protein toxins possessing similar properties and virulence (Casman,

While SAG must first bind to MHC class II molecules, their ability to activate large populations of T cells depends largely upon their binding to the TCR (Alber et al., 1990; Pontzer, et al., 199; Stevens, 1997; White et al., 1989). There are five different elements (Vα, Jα, Vβ, Dβ, and Jβ) that comprise the two-chain TCR receptor with each composed of a constant and variable region. The TCR uses all five elements for recognition of processed peptides on the surface of APCs (from conventional antigens). Peptide binding occurs within the peptide-binding region and requires a helper molecule such as CD4. Superantigens bind only to Vβ elements and specificity of binding results from the types of Vβ molecules present (Fig. 1). For example, SEB binds to human Vβ phenotypes 3, 12, 14, 15, 17, which differ from phenotypes bound by other SAGs (Table 2). Because many T cells contain the same Vβ phenotype, SAGs may activate up to 20% of the whole T-cell

SAGs are not restricted and can bind to both CD4 and CD8 T cells if the SAG recognizes the TCR Vβ chains, which increases the number of T cells they can affect. Superantigenactivated T cells can undergo at least five to six rounds of cell division (Nagshima et al., 2004). The massive clonal T-cell expansion results in the activation of programmed cell death in which cells responding to the specific superantigen are deleted (Choi et al., 1989; Yuh et al., 1993). Apoptosis of large numbers of T cells, clonal deletion and anergy of specific T-cell populations, and massive release of proinflammatory cytokines are all factors

There are two major diseases caused by the staphylococcal enterotoxin superantigens (SEs), and most diseases attributed to the SEs relate to chronic disease states caused by autoimmunity and repetitious stimulation by SEs. Those autoimmune diseases in which SEs are thought to play a role require more than one challenge and therefore, will probably not be a concern in a bioterrorism threat. The two diseases that are pertinent to potential bioterrorism attacks are SEB (or SEs) in the food or water supply (food poisoning) or an aerosol attack in which the toxin will be inhaled into the lungs, possibly causing toxic shock

As noted previously, *S. aureus* is a ubiquitous microorganism with a world-wide distribution, and is responsible for causing large numbers of food poisoning cases throughout the world (Bergdoll, 1989; Le Loir et al., 2003; Ortega, et al., 2010). In its normal environment, the gram-positive cocci and its toxins do not cause disease; however, when introduced into foods such as cream, mayonnaise, or similar foods, the bacteria grow rapidly secreting the exotoxins which then contaminate the food. Dack and coworkers (1930) provided the first documented report that identified a toxin from *S. aureus* as a causative agent of a food poisoning incident involving staphylococci-contaminated Christmas cake. The investigators grew the bacteria isolated from the cake and found that a sterile filtrate from the broth in which the bacteria were grown induced food poisoning when ingested by human volunteers. Thereafter, from investigations of various food poisoning outbreaks, an initial five antigenically distinct enterotoxins were identified suggesting that *S aureus* produced a family of protein toxins possessing similar properties and virulence (Casman,

population rather than the 0.01% activation by conventional antigens.

in the toxin's pathogenesis.

**4. SE pathogenesis** 

syndrome.

**4.1 Food poisoning** 

1960; Bergdoll et al., 1959; Bergdoll et al., 1965; Casman et al., 1967). Since the characterization of the five serotypes, at least 20 more SEs (Table 1) have been isolated and characterized with many inducing emesis in monkeys or humans (Uchiyama et al., 2003).


Table 2. Staphylococcal enterotoxins showing the corresponding TCR V repertoires

Approximately 25% of healthy people and animals carry *S. aureus* on the skin and often food workers who carry the bacterium may contaminate food when they handle food without washing their hands or wearing gloves. The microorganism is also found in unpasteurized milk and cheese products and, being salt tolerant, grows in salty foods as well. Because they are highly resistant to heat and enzymatic inactivation, foods that do not require cooking or those prepared by hand provide greater risks of contamination with the bacteria and subsequent toxin production. The short incubation period, approximately 4-6 hr after ingestion, usually differentiates SE-induced food poisoning from those caused by bacteria such as *E. coli* or *Salmonella* species where presence of the bacteria is required for disease.

Staphylococcal Enterotoxins, Stayphylococcal Enterotoxin B and Bioterrorism 49

leading cause of menstrual TSS (Chesney et al, 1981; Brosnanhan and Schlievert, 2010). TSST-1 is also a major etiologic agent for the other TSS category, non-menstrual TSS, comprising 50% of the reported cases (Buchdahl et al, 1985). Two other SEs, SEB and SEC, have been identified as the causative agent in most of the remaining 50%. Since its discovery, TSS had been considered a rare, but often fatal disease. After removal of absorbent tampons from the market and efforts to inform the public about TSS, incidences of menstrual TSS dropped from 13.7% to 0.3% per 100,000 individuals in the U. S. Since 1986, reported incidences of TSS in the U.S. have remained stable with the annual incidence rate around 0.32%-0.52% per 100,000 people (DeVries, et al., 2011; Haijeh et al., 1996). Several reasons may account for the disease's rarity. One reason lies in the fact that most adults have been exposed to SEs over a period of many years, and therefore, possesses antibodies against many of the SEs. The lack of anti-SE antibodies could also explain why children and young adults are more susceptible to the disease. In addition to immunity against SEs, another problem in TSS diagnosis relates to the lack of a single diagnostic test and, therefore, diagnosis relies upon a complex analysis of clinical symptoms. Requirements for a case to be identified as TSS are rigorous, and while SEs are significant virulence factors in many infections, most infections do not meet the diagnostic criteria for TSS as established

TSS pathophysiology involves many intricate extracellular and intracellular signaling pathways and at this time, the exact pathways or pathways responsible for the syndrome are not known (Davis et al., 1980; Kumar et al., 2010; Pinchuk et al., 2010). Hallmark studies during the 1990s showed that the toxicity of SEB was due to massive T-cell proliferation and proinflammatory cytokine production (Marrack et al., 1990; White et al., 1989). Investigations using T-cell reconstituted immuno-deficient SCID mice confirmed the role of T-cell activation in SE-induced lethality (Miethke et al., 1992; Canaan, et al. 1999). Furthermore, these studies indicated that tumor necrosis factor alpha (TNF-α) plays a crucial role in SE-induced lethality because passive immunization with an anti-TNF-α/β monoclonal antibody protected the animals against an SE challenge (Fast et al., 1989; Miethke et al., 1992). Although the precise mechanisms by which proinflammatory cytokines induce TSS, these early studies and further investigations provide overwhelming evidence that tissue damage, shock, and multiple organ failure is caused by the production of pathological concentrations of proinflammatory cytokines and chemokines such as TNFα, interleukin 1β (IL-1 β), interferon gamma (IFN-γ) and interleukin 6 (IL-6), and macrophage chemoattractant protein-1 (MCP-1) (Marrack et al., 1990; Williams, 1991).

Although T cells appear to play a dominant role in TSS, more recent investigations indicate that SE interactions with other cell types may also contribute to TSS pathophysiology (Das, 2000; Faulkner et al., 1997; Marrack et al., 1990; McCormick et al. 2001). Initially, the purpose of the APC MHC class II interaction with SEs was thought to provide a mechanism for T-cell activation and production of proinflammatory cytokines by T-cell populations. That MHC class II interactions play a more active role in TSS was shown by studies in which mortality from TSST-1 was not reduced in T-cell depleted rabbits (Dinges et al, 2003). In addition, TSST-1 induced proinflammatory cytokines in these animals, again suggesting that other cells may play a role in cytokine production. These studies are further supported by investigations in which the SE-MHC class II interaction by itself was shown to be sufficient to activate intracellular signaling pathways which induce downstream pro-inflammatory signaling and

subsequent production of cytokines (Kisner (a) et al., 2011; Kisner (b) et al., 2011).

by CDC (Table 3).

The onset is sudden and vomiting is the hallmark symptom. Other symptoms, such as diarrhea, abdominal pain, and nausea may also be present, but systemic manifestations such as fever are very uncommon (Alouf and Muller-Alouf, 2003; Kerouanton et al., 2007). Although extremely incapacitating, staphylococcal food poisoning is usually self-limiting and symptoms last about 1 day. Fatality in healthy adults is rare (0.03%); however, the rate is higher in susceptible populations (children, elderly, and immune-compromised adults) and may also depend upon the concentration of the toxin ingested (Do Carmo, et al., 2004).

SE-induced food poisoning was initially thought to be caused by the local interaction of the toxin with intestinal cells because the toxin stimulates nerve centers in the gut through serotonin (5-hydroxytryptamine or 5 HT) release from intestinal mast cells (Alouf and Muller-Alouf, 2003; Hu et al., 2007). Serotonin binds to 5-HT3 receptors which are ligand-gated ion channels located on the afferent vagus nerve terminals. The binding of serotonin to the receptor opens the channel which signals the medulla emetic reflex center to generate nausea and an emetic response. However, such interactions do not explain the disease pathophysiology. Patients with SE intoxication can exhibit rather severe gastrointestinal damage including mucosal hyperemia, regional edema, petechiae, and purulent exudates (Ortega et al., 2010; Palmer, 1951). SEs cross the intestinal epithelial barrier and gain access to local and systemic lymphoid tissues, suggesting that activation of local immune tissue may be partially responsible for gastrointestinal damage (Hamad et al., 1997). Involvement of the immune system in pathogenesis could explain why immuno-compromised adults develop a more severe, life-threatening disease than normal healthy adults. However, emesis is not directly linked to T-cell proliferation because TSST-1, a potent immune activator, does not cause emesis. TSST-1 is more susceptible to enzymes in the digestive tract and could be the reason for its lack of emetic activity. SEs with emetic activity have a disulfide bond located at the top of the B domain and probably are responsible for stabilizing the molecule in a conformation needed to induce emesis (Brosnahan and Schievert, 20l1).

#### **4.2 Toxic shock syndrome**

During the late 1970's, Todd and coworkers (1978) described an acute illness in seven children, between the ages of 8 to 17. Symptoms included high fever, hypotension, vomiting, watery diarrhea, a scarlatiniform rash, and renal failure. Although bacteria were not isolated from blood, cerebrospinal fluid, or urine, S. aureus was isolated from mucosal sites. Culture filtrates from cultures of these isolates were shown to contain a toxin that would cause a rash (Nikolksy sign) in newborn mice. Todd named the new disease toxic shock syndrome (TSS). In 1977 through 1980, 22 women between the ages of 13-24, were diagnosed with TSS (Chesney et al., 1981). Investigations showed that this TSS was the result of highly absorbent tampons, which became contaminated with *S. aureus*. A toxin, tsst-1, was isolated from the bacteria cultured from the contaminated tampon and shown to be similar to that discovered by Todd et al. (1978). The super absorbent tampons were found to introduce oxygen into the anaerobic environment of the vagina which facilitated the growth of *S. aureus* and release of TSST-1. When the absorbent tampons were taken off the market, the incidence of menstrual TSS decreased dramatically.

From these early investigations, TSS has been divided into two categories. The first, menstrual TSS, occurs primarily in young women, ages 16-25, during menstrual periods and is usually associated with tampon use. As first identified in the 1980s, TSST-1, remains the

The onset is sudden and vomiting is the hallmark symptom. Other symptoms, such as diarrhea, abdominal pain, and nausea may also be present, but systemic manifestations such as fever are very uncommon (Alouf and Muller-Alouf, 2003; Kerouanton et al., 2007). Although extremely incapacitating, staphylococcal food poisoning is usually self-limiting and symptoms last about 1 day. Fatality in healthy adults is rare (0.03%); however, the rate is higher in susceptible populations (children, elderly, and immune-compromised adults) and may also depend upon the concentration of the toxin ingested (Do Carmo, et al., 2004). SE-induced food poisoning was initially thought to be caused by the local interaction of the toxin with intestinal cells because the toxin stimulates nerve centers in the gut through serotonin (5-hydroxytryptamine or 5 HT) release from intestinal mast cells (Alouf and Muller-Alouf, 2003; Hu et al., 2007). Serotonin binds to 5-HT3 receptors which are ligand-gated ion channels located on the afferent vagus nerve terminals. The binding of serotonin to the receptor opens the channel which signals the medulla emetic reflex center to generate nausea and an emetic response. However, such interactions do not explain the disease pathophysiology. Patients with SE intoxication can exhibit rather severe gastrointestinal damage including mucosal hyperemia, regional edema, petechiae, and purulent exudates (Ortega et al., 2010; Palmer, 1951). SEs cross the intestinal epithelial barrier and gain access to local and systemic lymphoid tissues, suggesting that activation of local immune tissue may be partially responsible for gastrointestinal damage (Hamad et al., 1997). Involvement of the immune system in pathogenesis could explain why immuno-compromised adults develop a more severe, life-threatening disease than normal healthy adults. However, emesis is not directly linked to T-cell proliferation because TSST-1, a potent immune activator, does not cause emesis. TSST-1 is more susceptible to enzymes in the digestive tract and could be the reason for its lack of emetic activity. SEs with emetic activity have a disulfide bond located at the top of the B domain and probably are responsible for stabilizing the molecule in a

conformation needed to induce emesis (Brosnahan and Schievert, 20l1).

market, the incidence of menstrual TSS decreased dramatically.

During the late 1970's, Todd and coworkers (1978) described an acute illness in seven children, between the ages of 8 to 17. Symptoms included high fever, hypotension, vomiting, watery diarrhea, a scarlatiniform rash, and renal failure. Although bacteria were not isolated from blood, cerebrospinal fluid, or urine, S. aureus was isolated from mucosal sites. Culture filtrates from cultures of these isolates were shown to contain a toxin that would cause a rash (Nikolksy sign) in newborn mice. Todd named the new disease toxic shock syndrome (TSS). In 1977 through 1980, 22 women between the ages of 13-24, were diagnosed with TSS (Chesney et al., 1981). Investigations showed that this TSS was the result of highly absorbent tampons, which became contaminated with *S. aureus*. A toxin, tsst-1, was isolated from the bacteria cultured from the contaminated tampon and shown to be similar to that discovered by Todd et al. (1978). The super absorbent tampons were found to introduce oxygen into the anaerobic environment of the vagina which facilitated the growth of *S. aureus* and release of TSST-1. When the absorbent tampons were taken off the

From these early investigations, TSS has been divided into two categories. The first, menstrual TSS, occurs primarily in young women, ages 16-25, during menstrual periods and is usually associated with tampon use. As first identified in the 1980s, TSST-1, remains the

**4.2 Toxic shock syndrome** 

leading cause of menstrual TSS (Chesney et al, 1981; Brosnanhan and Schlievert, 2010). TSST-1 is also a major etiologic agent for the other TSS category, non-menstrual TSS, comprising 50% of the reported cases (Buchdahl et al, 1985). Two other SEs, SEB and SEC, have been identified as the causative agent in most of the remaining 50%. Since its discovery, TSS had been considered a rare, but often fatal disease. After removal of absorbent tampons from the market and efforts to inform the public about TSS, incidences of menstrual TSS dropped from 13.7% to 0.3% per 100,000 individuals in the U. S. Since 1986, reported incidences of TSS in the U.S. have remained stable with the annual incidence rate around 0.32%-0.52% per 100,000 people (DeVries, et al., 2011; Haijeh et al., 1996). Several reasons may account for the disease's rarity. One reason lies in the fact that most adults have been exposed to SEs over a period of many years, and therefore, possesses antibodies against many of the SEs. The lack of anti-SE antibodies could also explain why children and young adults are more susceptible to the disease. In addition to immunity against SEs, another problem in TSS diagnosis relates to the lack of a single diagnostic test and, therefore, diagnosis relies upon a complex analysis of clinical symptoms. Requirements for a case to be identified as TSS are rigorous, and while SEs are significant virulence factors in many infections, most infections do not meet the diagnostic criteria for TSS as established by CDC (Table 3).

TSS pathophysiology involves many intricate extracellular and intracellular signaling pathways and at this time, the exact pathways or pathways responsible for the syndrome are not known (Davis et al., 1980; Kumar et al., 2010; Pinchuk et al., 2010). Hallmark studies during the 1990s showed that the toxicity of SEB was due to massive T-cell proliferation and proinflammatory cytokine production (Marrack et al., 1990; White et al., 1989). Investigations using T-cell reconstituted immuno-deficient SCID mice confirmed the role of T-cell activation in SE-induced lethality (Miethke et al., 1992; Canaan, et al. 1999). Furthermore, these studies indicated that tumor necrosis factor alpha (TNF-α) plays a crucial role in SE-induced lethality because passive immunization with an anti-TNF-α/β monoclonal antibody protected the animals against an SE challenge (Fast et al., 1989; Miethke et al., 1992). Although the precise mechanisms by which proinflammatory cytokines induce TSS, these early studies and further investigations provide overwhelming evidence that tissue damage, shock, and multiple organ failure is caused by the production of pathological concentrations of proinflammatory cytokines and chemokines such as TNFα, interleukin 1β (IL-1 β), interferon gamma (IFN-γ) and interleukin 6 (IL-6), and macrophage chemoattractant protein-1 (MCP-1) (Marrack et al., 1990; Williams, 1991).

Although T cells appear to play a dominant role in TSS, more recent investigations indicate that SE interactions with other cell types may also contribute to TSS pathophysiology (Das, 2000; Faulkner et al., 1997; Marrack et al., 1990; McCormick et al. 2001). Initially, the purpose of the APC MHC class II interaction with SEs was thought to provide a mechanism for T-cell activation and production of proinflammatory cytokines by T-cell populations. That MHC class II interactions play a more active role in TSS was shown by studies in which mortality from TSST-1 was not reduced in T-cell depleted rabbits (Dinges et al, 2003). In addition, TSST-1 induced proinflammatory cytokines in these animals, again suggesting that other cells may play a role in cytokine production. These studies are further supported by investigations in which the SE-MHC class II interaction by itself was shown to be sufficient to activate intracellular signaling pathways which induce downstream pro-inflammatory signaling and subsequent production of cytokines (Kisner (a) et al., 2011; Kisner (b) et al., 2011).

Staphylococcal Enterotoxins, Stayphylococcal Enterotoxin B and Bioterrorism 51

SEB interacts with (A) lymphoid cells and induces production of proinflammatory cytokines. SEB stimulates (B) endothelial and (C) epithelial cells to release cytokines (some proinflammatory)/chemokines and growth factors. Red arrows show interactions between cell types are also affected by SEB-induced release of these factors that ultimately leads to endothelial cell dysfunction, vascular leak, and shock resulting from multi-organ failure.

SE binding to lymphoid cells has been extensively characterized and interactions of SEs with these cells are fairly well understood (Achara and Baker, 2004; Broshanan et al. 2011; Larkin et al., 2009). The complexity of the disease suggests that SEs may affect more than lymphoid cells, and recently, an epithelial binding moiety has been identified on the SE molecule (Brosnahan and Schlievert, 20011). SEs bind to epithelial cells and elicit the production of specific cytokines. SEs have also been shown to cross polarized epithelial cells *in vitro* suggesting that SEs gain systemic access to the body (Hale, unpublished data). Rajagpjalan et al. (2007) show acute activation of the systemic immune system and inflammatory response in mice that were vaginally or intranasally exposed to SEB (Rajagopalan et al. 2007; Rajagopalan et al., 2006). Since the toxin was introduced on mucosal surfaces, the only method for systemic activation would be if the toxin crossed the epithelial cell barrier and gained access to the body. Vaginal epithelial cells were also shown to bind TSST-1 and induce TNFα production while treatment of epithelial cells with TSST-1 or SEB induced

SE-induced shock causes severe damage to the endothelial vasculature and vascular leak contributes significantly to TSS pathology (Krakauer, 1994; Ortega et al. 2010). Elevated levels of vascular endothelial growth factor are observed in serum from patients with sepsis

production of MIP-3α and IL-8 (Brosnahan et al., 2001; Peterson, et al., 2005).

Fig. 3. Systemic SEB (SE) intoxication is a complex disease

#### **Clinical case definition**


#### **Multisystem involvement (three or more of the following organ systems:**

•Gastrointestinal: vomiting or diarrhea at onset of illness

•Muscular: severe myalgia or creatine phosphokinase level

at least twice the upper limit of normal

•Mucous membrane: vaginal, oropharyngeal, or conjunctival hyperemia

•Renal: blood urea nitrogen or creatinine at least twice the upper limit of normal for laboratory or tract infection

•Urinary sediment with pyuria (greater than or equal to 5 leukocytes

per high-power field) in the absence of urinary tract infections

•Hepatic: total bilirubin, alanine aminotransferase enzyme, or asparate

aminotransferase enzyme levels at least twice the upper limit of normal values

•Hematologic: platelets less than 100,000/mm3

•Central nervous system: disorientation or alterations in consciousness without focal neurologic signs when fever and hypotension are absent

#### **Laboratory criteria for diagnosis**

•Negative results on the following tests, if obtained:

•Blood or cerebrospinal fluid cultures blood culture may be positive for

*Staphylococcus aureus* 

•Negative serologies for Rocky Mountain spotted fever, leptospirosis, or measles

#### **Case classification**

•Probable: a case which meets the laboratory criteria and in which four of the five clinical findings described above are present

•Confirmed: a case which meets the laboratory criteria and in which all five of the clinical findings described above are present, including desquamation,

unless the patient dies before desquamation occurs

CSTE Position Statement Number: 10-ID-14

Table 3. CDC 1997 Definition for Toxic Shock Syndrome

•Hypotension: systolic blood pressure less than or equal to 90 mm Hg

**Multisystem involvement (three or more of the following organ systems:** 

•Mucous membrane: vaginal, oropharyngeal, or conjunctival hyperemia •Renal: blood urea nitrogen or creatinine at least twice the upper limit of

•Urinary sediment with pyuria (greater than or equal to 5 leukocytes

•Hepatic: total bilirubin, alanine aminotransferase enzyme, or asparate

•Central nervous system: disorientation or alterations in consciousness without focal neurologic signs when fever and hypotension are absent

•Blood or cerebrospinal fluid cultures blood culture may be positive for

•Negative serologies for Rocky Mountain spotted fever, leptospirosis,

•Probable: a case which meets the laboratory criteria and in which four of the

•Confirmed: a case which meets the laboratory criteria and in which all five of the clinical findings described above are present, including desquamation,

aminotransferase enzyme levels at least twice the upper limit of normal values

per high-power field) in the absence of urinary tract infections

for adults or less than fifth percentile by age for children aged less than 16 years

• Fever: temperature greater than or equal to 38.9°C

•Gastrointestinal: vomiting or diarrhea at onset of illness •Muscular: severe myalgia or creatine phosphokinase level

•Rash: diffuse macular erythroderma

**Clinical case definition**

at least twice the upper limit of normal

normal for laboratory or tract infection

**Laboratory criteria for diagnosis**

*Staphylococcus aureus* 

**Case classification**

or measles

•Hematologic: platelets less than 100,000/mm3

•Negative results on the following tests, if obtained:

five clinical findings described above are present

unless the patient dies before desquamation occurs

CSTE Position Statement Number: 10-ID-14

Table 3. CDC 1997 Definition for Toxic Shock Syndrome

•Desquamation: 1-2 weeks after onset of rash

Fig. 3. Systemic SEB (SE) intoxication is a complex disease

SEB interacts with (A) lymphoid cells and induces production of proinflammatory cytokines. SEB stimulates (B) endothelial and (C) epithelial cells to release cytokines (some proinflammatory)/chemokines and growth factors. Red arrows show interactions between cell types are also affected by SEB-induced release of these factors that ultimately leads to endothelial cell dysfunction, vascular leak, and shock resulting from multi-organ failure.

SE binding to lymphoid cells has been extensively characterized and interactions of SEs with these cells are fairly well understood (Achara and Baker, 2004; Broshanan et al. 2011; Larkin et al., 2009). The complexity of the disease suggests that SEs may affect more than lymphoid cells, and recently, an epithelial binding moiety has been identified on the SE molecule (Brosnahan and Schlievert, 20011). SEs bind to epithelial cells and elicit the production of specific cytokines. SEs have also been shown to cross polarized epithelial cells *in vitro* suggesting that SEs gain systemic access to the body (Hale, unpublished data). Rajagpjalan et al. (2007) show acute activation of the systemic immune system and inflammatory response in mice that were vaginally or intranasally exposed to SEB (Rajagopalan et al. 2007; Rajagopalan et al., 2006). Since the toxin was introduced on mucosal surfaces, the only method for systemic activation would be if the toxin crossed the epithelial cell barrier and gained access to the body. Vaginal epithelial cells were also shown to bind TSST-1 and induce TNFα production while treatment of epithelial cells with TSST-1 or SEB induced production of MIP-3α and IL-8 (Brosnahan et al., 2001; Peterson, et al., 2005).

SE-induced shock causes severe damage to the endothelial vasculature and vascular leak contributes significantly to TSS pathology (Krakauer, 1994; Ortega et al. 2010). Elevated levels of vascular endothelial growth factor are observed in serum from patients with sepsis

Staphylococcal Enterotoxins, Stayphylococcal Enterotoxin B and Bioterrorism 53

for studying TSS, but each model has its own limitations, which need to be understood in

The mouse remains the most common model for TSS studies, although they are not sensitive to the toxin and must be sensitized with either hepatotoxins (e.g., D-galactosamine and actinomycin D) or with endotoxin to achieve an effect (Chen et al., 1994; Nagaki et al., 1994; Blank et al., 1997; Sugiyama et al., 1964). Endotoxin is a natural component of gram-negative bacteria found in the intestines and may actually contribute to shock syndromes. Although tissue damage from SE and lipopolysaccharide (LPS) may vary, acute shock caused by abnormally high levels of TNF-α and other proinflammatory cytokines results in lifethreatening situations (Das, 2000; Miekthe et al, 1997; Sifka and Whitton, 2000). Thus, an animal model in which the SEB effects are magnified by sublethal concentrations of LPS provides an *in vivo* system useful for studying various facets of lethal shock. While each mouse model lacks some characteristics of the disease in humans, Krakauer et al. (2010) found that three different mouse models with different susceptibility to SEB could be used

T-cell deficient mice or mice engineered to have specific cytokine deficiencies show that TNF-α and T cells are both required for SE-induced lethality (Blank, et al. 1997). Transgenic mice expressing human TCR/MHC class II determinants solve some problems associated with mouse lymphoid cells binding SE and SAG-sensitive mice show a biphasic release of cytokines with early TNF-a release mediating lethal shock (Faulkner et al., 2005; Rajagopalan et al., 2002). These investigations point also to the spleen as a major source of TNF-a production during an acute (early) cytokine response. The studies support the idea that TSS is not simply due to cytokines released by T cells, but entails a series of events affecting major organs throughout the body. Recently, a humanized mouse in which T-cell immune deficient mice, SCID, were transfused with human hematopoietic fetal liver CD34+ cells that had previously been implanted with human fetal thymic and liver tissues developed long-term human innate and adaptive immune responses. When TSST-1 was injected into these mice, the mice responded immunologically in a manner similar to humans (Melkus et al., 2006) suggesting that this mouse model may overcome many of the

Rats have been an excellent model to study TSS effects on the nervous system. Wang et al, (2004) showed activation of neuronal developmental genes after rats were given intraperitoneal injections of SEB. Activation appeared to occur through the tenth cranial nerve, the vagus, because severing this nerve prevented neuronal activation. These studies support the idea that brain-immune system communications play a role in TSS. Some sequalae of TSS relate to memory loss and confusion which would indicate involvement of

Because a major drawback for murine and rodent models in TSS is the lack of clinical symptoms that occur in humans, a porcine model has been developed in which 18-day-old

problems associated with mouse models for SE intoxication.

the nervous system (Kusnocov and Goldfaith, 2005).

order to use each model in furthering our understanding of the disease process.

**5.1 Mouse** 

to study SEB intoxication.

**5.2 Rat** 

**5.3 Minature swine** 

or septic shock (Karlsson et al. 2008). Because there are common features among hemorrhagic shock, septic shock, and toxic shock syndrome, factors that regulate endothelial homeostasis are probably important in its prevention. Future studies examining the interplay among lymphoid, endothelial and epithelial cells will provide more understanding of the disease and enable a logical approach for therapy.

#### **4.3 Pulmonary complications**

One of the most effective and deadly forms of a bioterrorism attack is delivery of the toxin or microorganism by aerosol exposure (Ulrich et al., 1997). Understanding SE-intoxication in humans has been difficult because there is no direct comparison between the pathogenesis of human disease and the disease caused by an intentional aerosol attack. Perhaps the most descriptive and informative reports detailed an accidental laboratory inhalational exposure of fifteen workers (Rusnak et al., 2004). Ten became symptomatic and nine were hospitalized. The onset was rapid (1 1/2 hrs to 24 hr) after exposure with the illness lasting 3-4 days. Commonly observed symptoms were fever, headache, myalgias, pulmonary symptoms, and gastrointestinal symptoms.

A Rhesus macaque animal model was used to characterize SEB intoxication by an aerosol route. In these studies, nonhuman primates (NHP) were exposed to a lethal dose (5 LD50) of aerosolized SEB in a modified Henderson head-only aerosol exposure chamber. NHPs developed gastrointestinal symptoms (anorexia, diarrhea, and emesis) within 24 hr after exposure. The gastrointestinal symptoms appeared to be self-limiting, but 24 hr later, the NHPs developed an abrupt onset of lethargy, dyspnea, and facial pallor. Usually within 4 hr, the animals died or were euthanized when moribund. Postmortem examination revealed lesions in the lungs and signs of pulmonary edema. Both large and small intestines showed petechial hemorrhaging and mucosal erosion, and lymph nodes were swollen. There was definite damage to the endothelium and endothelial cells. The authors of the study concluded that SEB is a potent stimulant in rhesus monkeys and that a similar dose in humans could produce similar symptoms. One thing to consider, however, when extrapolating from NHP to human, is that the NHP were seronegative when tested for the presence of antibodies against SEB; most humans have some degree of past exposure to the toxin and therefore would perhaps have some immunity.

Since these studies on NHP, there is evidence that links SEs exposure to asthma and respiratory problems (Kumar et al., 2010). Inflammatory reactions in the lung are induced by TNF-α and two life-threatening syndromes, vascular leak and respiratory distress develop during toxic shock (Aubert et al., 2000; Herz et al., 1999; Neuman et al., 1997). Other studies show that cytokine-mediated acute respiratory distress syndrome and inflammatory lung disease occur during SE intoxication (DeSouza et al., 2006; Fujisawa et al., 1998; Slifka and Whitton, 2002). Both conditions are critical and may be lethal without therapeutic intervention (Das, 2000; Kasai et al., 1997). Anti-inflammatory drugs reduce TNF-α and other proinflammatory cytokines which alleviates the symptoms and helps the individuals to recover from these life-threatening illnesses.

#### **5. Animal models**

A major problem in understanding the disease process of SE intoxication is the lack of appropriate animal models that mimic the human disease. There are several animal models for studying TSS, but each model has its own limitations, which need to be understood in order to use each model in furthering our understanding of the disease process.

#### **5.1 Mouse**

52 Bioterrorism

or septic shock (Karlsson et al. 2008). Because there are common features among hemorrhagic shock, septic shock, and toxic shock syndrome, factors that regulate endothelial homeostasis are probably important in its prevention. Future studies examining the interplay among lymphoid, endothelial and epithelial cells will provide more

One of the most effective and deadly forms of a bioterrorism attack is delivery of the toxin or microorganism by aerosol exposure (Ulrich et al., 1997). Understanding SE-intoxication in humans has been difficult because there is no direct comparison between the pathogenesis of human disease and the disease caused by an intentional aerosol attack. Perhaps the most descriptive and informative reports detailed an accidental laboratory inhalational exposure of fifteen workers (Rusnak et al., 2004). Ten became symptomatic and nine were hospitalized. The onset was rapid (1 1/2 hrs to 24 hr) after exposure with the illness lasting 3-4 days. Commonly observed symptoms were fever, headache, myalgias, pulmonary

A Rhesus macaque animal model was used to characterize SEB intoxication by an aerosol route. In these studies, nonhuman primates (NHP) were exposed to a lethal dose (5 LD50) of aerosolized SEB in a modified Henderson head-only aerosol exposure chamber. NHPs developed gastrointestinal symptoms (anorexia, diarrhea, and emesis) within 24 hr after exposure. The gastrointestinal symptoms appeared to be self-limiting, but 24 hr later, the NHPs developed an abrupt onset of lethargy, dyspnea, and facial pallor. Usually within 4 hr, the animals died or were euthanized when moribund. Postmortem examination revealed lesions in the lungs and signs of pulmonary edema. Both large and small intestines showed petechial hemorrhaging and mucosal erosion, and lymph nodes were swollen. There was definite damage to the endothelium and endothelial cells. The authors of the study concluded that SEB is a potent stimulant in rhesus monkeys and that a similar dose in humans could produce similar symptoms. One thing to consider, however, when extrapolating from NHP to human, is that the NHP were seronegative when tested for the presence of antibodies against SEB; most humans have some degree of past exposure to the

Since these studies on NHP, there is evidence that links SEs exposure to asthma and respiratory problems (Kumar et al., 2010). Inflammatory reactions in the lung are induced by TNF-α and two life-threatening syndromes, vascular leak and respiratory distress develop during toxic shock (Aubert et al., 2000; Herz et al., 1999; Neuman et al., 1997). Other studies show that cytokine-mediated acute respiratory distress syndrome and inflammatory lung disease occur during SE intoxication (DeSouza et al., 2006; Fujisawa et al., 1998; Slifka and Whitton, 2002). Both conditions are critical and may be lethal without therapeutic intervention (Das, 2000; Kasai et al., 1997). Anti-inflammatory drugs reduce TNF-α and other proinflammatory cytokines which alleviates the symptoms and helps the individuals

A major problem in understanding the disease process of SE intoxication is the lack of appropriate animal models that mimic the human disease. There are several animal models

understanding of the disease and enable a logical approach for therapy.

**4.3 Pulmonary complications** 

symptoms, and gastrointestinal symptoms.

toxin and therefore would perhaps have some immunity.

to recover from these life-threatening illnesses.

**5. Animal models** 

The mouse remains the most common model for TSS studies, although they are not sensitive to the toxin and must be sensitized with either hepatotoxins (e.g., D-galactosamine and actinomycin D) or with endotoxin to achieve an effect (Chen et al., 1994; Nagaki et al., 1994; Blank et al., 1997; Sugiyama et al., 1964). Endotoxin is a natural component of gram-negative bacteria found in the intestines and may actually contribute to shock syndromes. Although tissue damage from SE and lipopolysaccharide (LPS) may vary, acute shock caused by abnormally high levels of TNF-α and other proinflammatory cytokines results in lifethreatening situations (Das, 2000; Miekthe et al, 1997; Sifka and Whitton, 2000). Thus, an animal model in which the SEB effects are magnified by sublethal concentrations of LPS provides an *in vivo* system useful for studying various facets of lethal shock. While each mouse model lacks some characteristics of the disease in humans, Krakauer et al. (2010) found that three different mouse models with different susceptibility to SEB could be used to study SEB intoxication.

T-cell deficient mice or mice engineered to have specific cytokine deficiencies show that TNF-α and T cells are both required for SE-induced lethality (Blank, et al. 1997). Transgenic mice expressing human TCR/MHC class II determinants solve some problems associated with mouse lymphoid cells binding SE and SAG-sensitive mice show a biphasic release of cytokines with early TNF-a release mediating lethal shock (Faulkner et al., 2005; Rajagopalan et al., 2002). These investigations point also to the spleen as a major source of TNF-a production during an acute (early) cytokine response. The studies support the idea that TSS is not simply due to cytokines released by T cells, but entails a series of events affecting major organs throughout the body. Recently, a humanized mouse in which T-cell immune deficient mice, SCID, were transfused with human hematopoietic fetal liver CD34+ cells that had previously been implanted with human fetal thymic and liver tissues developed long-term human innate and adaptive immune responses. When TSST-1 was injected into these mice, the mice responded immunologically in a manner similar to humans (Melkus et al., 2006) suggesting that this mouse model may overcome many of the problems associated with mouse models for SE intoxication.

#### **5.2 Rat**

Rats have been an excellent model to study TSS effects on the nervous system. Wang et al, (2004) showed activation of neuronal developmental genes after rats were given intraperitoneal injections of SEB. Activation appeared to occur through the tenth cranial nerve, the vagus, because severing this nerve prevented neuronal activation. These studies support the idea that brain-immune system communications play a role in TSS. Some sequalae of TSS relate to memory loss and confusion which would indicate involvement of the nervous system (Kusnocov and Goldfaith, 2005).

#### **5.3 Minature swine**

Because a major drawback for murine and rodent models in TSS is the lack of clinical symptoms that occur in humans, a porcine model has been developed in which 18-day-old

Staphylococcal Enterotoxins, Stayphylococcal Enterotoxin B and Bioterrorism 55

homeostasis. Recommendations for treating TSS include first the removal of any foreign materials that might be contaminated with *S aureus* ( i.e., tampons or nasal packings) and draining sites of infection to prevent further bacterial growth. Treatment with antimicrobials is also recommended if sepsis is involved. In severe cases, other therapeutic interventions that may minimize the risk of tissue damage and organ failure include fluids to prevent dehydration, dialysis if severe kidney problems occur, drugs to control blood pressure and cardiovascular function, anti-inflammatory agents, and possibly insulin, if needed. Supportive care should be aggressive and monitored carefully. Length of

As shown by studies in NHP, animals could be protected against SE intoxication using passive immunization with anti-SEB antibodies. The antibodies provided protection if given up to 4 hr after the NHPs had received 5 LD50sof aerosolized SEB (LeClaire et al., 2002). These studies showed that the antibodies were able to neutralize the toxin *in vivo* and provide protection after intoxication had occurred. Unfortunately, the antibody was a monoclonal antibody (Mab) created in a chicken, and the antibody itself may be antigenic and cause serum sickness. There are several anti-SEB Mab preparations that will not cause adverse reactions in humans. Because most Mabs are not developed using human cells, Mab development for human use will require "humanization of the Mab in order to eliminate the molecule's antigenicity (Goldsmith and Signore, 2010). Investigations using immune lymphocytes to prepare Mabs may provide therapeutics without the expense of humanization. Additionally, developing small-domain antibody fragments such as camilid antibodies may also provide therapeutic agents that, because of their size, may not be antigenic in humans (Graef et al., 2011). Further studies are needed to identify antibody reagents that will be successful therapeutics, and whether the antibody reagents will need to

Over the past 20 years, there have been numerous investigations showing efficacy of various pharmacologic agents that prevent or delay lethality when animals are treated after a SE challenge, and as yet none has advanced to human clinical trials (Krakauer, 2010). Human activated protein C (hAPC) was approved for patients in severe septic shock, and had been used to treat TSS (DeVries et al. 2011). On October 25, 2011, Eli Lilly withdrew *Xigris (hAPC) because more in depth clinical trials indicated that drug did no better than a placebo in reducing mortality (*http://www.medscape.com/viewarticle/752169*).* The complexity of TSS tends to discount development of a single drug capable of treating the disease . Because the disease affects the endothelial cell vasculature of multiple organ systems, including drugs that treat

endothelial cell damage should also help to establish a therapeutic regimen for TSS.

To protect soldiers against SEB exposure, the U. S. Army developed a formalin-treated SEB toxoid that had some degree of success in protecting animals against a SEB challenge (Tseng et al., 1995). Due to the fact that formalin treatment did not always inactivate every toxin molecule, there was a move to develop vaccines without the need for formalin

hospitalization may vary between 5 and 11 days (DeVries et al., 2011).

be combined with pharmacologic agents to enhance their efficacy.

**6.3 Possible pharmacologic agents to treat SE intoxication** 

**6.4 Vaccines** 

**6.2 Antibody therapy for SE intoxication** 

piglets are given a lethal dose of SEB intravenously (van Gessel et al., 2004). Intoxicated piglets develop vomiting, diarrhea, febrile temperature spikes, anorexia, and hypotension similar to the clinical course of disease observed in humans and may offer an *in vivo* model with more of the symptoms observed in human TSS.

#### **5.4 Rabbit**

McCormick et al. (2003) noted that rabbits, when given SAG by a continuous perfusion, developed pyrogenic symptoms similar to humans. Investigations showed that the lethal pathology was similar to that observed in human TSS, but with newer animal models and a greater understanding of mechanisms involved in TSS, rabbits are not a major animal model for TSS.

#### **5.5 Shrew**

Most of the small animal models used to study the effects of SE-intoxication do not display an emetic response (King, 1990). Hu et al. (2003) developed an emetic model with which to study SE-induced emesis. They reported that several SE serotypes, known to induce emesis in NHP, induced emesis in the house musk shrew (*Suncus murinus*). Concentrations required to induce an emetic response were approximately 0.4 μg per animal, but the dose varied with the SE serotype. Variations in SE toxicity among the serotypes were similar to those observed in NPHs and humans, making the shrew an excellent small animal model to study emesis induced by SEs.

#### **5.6 Nonhuman primate**

NHP exhibit a similar disease progression as that observed in humans and are susceptible when given SEB orally (Boles et al., 2003; Mattix et al., 1995; Ulrich et al., 1997). Because there are limitations in the number of NHP available for studies and because of the expense involved in NHP studies, they are usually reserved for preclinical investigations. While TSS manifestations in NHPs resemble those observed in humans, there are differences between the immune systems, which may become more evident as more is learned about the disease process.

#### **6. Therapy and prophylaxis**

Most therapeutic and prophylatic measures are concerned with TSS or systemic SE intoxication because food poisoning is usually self-limiting. Although identification of unusual food poisoning incidents should be monitored as a possible biothreat action, the disease itself should not be life-threatening, and recovery occurs without serious side effects. Because therapy for food poisoning is not a serious concern, this section will address the measures to treat disease pathogenesis resulting from systemic SE intoxication.

#### **6.1 Current therapeutic measures**

At the current time, intravenous human gamma globulins (IVIG) is the primary therapeutic to treat TSS. Because there are no specific drugs available for treatment, a primary goal of any therapeutic intervention is to maintain important body functions and physiological homeostasis. Recommendations for treating TSS include first the removal of any foreign materials that might be contaminated with *S aureus* ( i.e., tampons or nasal packings) and draining sites of infection to prevent further bacterial growth. Treatment with antimicrobials is also recommended if sepsis is involved. In severe cases, other therapeutic interventions that may minimize the risk of tissue damage and organ failure include fluids to prevent dehydration, dialysis if severe kidney problems occur, drugs to control blood pressure and cardiovascular function, anti-inflammatory agents, and possibly insulin, if needed. Supportive care should be aggressive and monitored carefully. Length of hospitalization may vary between 5 and 11 days (DeVries et al., 2011).

#### **6.2 Antibody therapy for SE intoxication**

54 Bioterrorism

piglets are given a lethal dose of SEB intravenously (van Gessel et al., 2004). Intoxicated piglets develop vomiting, diarrhea, febrile temperature spikes, anorexia, and hypotension similar to the clinical course of disease observed in humans and may offer an *in vivo* model

McCormick et al. (2003) noted that rabbits, when given SAG by a continuous perfusion, developed pyrogenic symptoms similar to humans. Investigations showed that the lethal pathology was similar to that observed in human TSS, but with newer animal models and a greater understanding of mechanisms involved in TSS, rabbits are not a major animal model

Most of the small animal models used to study the effects of SE-intoxication do not display an emetic response (King, 1990). Hu et al. (2003) developed an emetic model with which to study SE-induced emesis. They reported that several SE serotypes, known to induce emesis in NHP, induced emesis in the house musk shrew (*Suncus murinus*). Concentrations required to induce an emetic response were approximately 0.4 μg per animal, but the dose varied with the SE serotype. Variations in SE toxicity among the serotypes were similar to those observed in NPHs and humans, making the shrew an excellent small animal model to

NHP exhibit a similar disease progression as that observed in humans and are susceptible when given SEB orally (Boles et al., 2003; Mattix et al., 1995; Ulrich et al., 1997). Because there are limitations in the number of NHP available for studies and because of the expense involved in NHP studies, they are usually reserved for preclinical investigations. While TSS manifestations in NHPs resemble those observed in humans, there are differences between the immune systems, which may become more evident as more is learned about the disease

Most therapeutic and prophylatic measures are concerned with TSS or systemic SE intoxication because food poisoning is usually self-limiting. Although identification of unusual food poisoning incidents should be monitored as a possible biothreat action, the disease itself should not be life-threatening, and recovery occurs without serious side effects. Because therapy for food poisoning is not a serious concern, this section will address the

At the current time, intravenous human gamma globulins (IVIG) is the primary therapeutic to treat TSS. Because there are no specific drugs available for treatment, a primary goal of any therapeutic intervention is to maintain important body functions and physiological

measures to treat disease pathogenesis resulting from systemic SE intoxication.

with more of the symptoms observed in human TSS.

**5.4 Rabbit** 

for TSS.

**5.5 Shrew** 

process.

study emesis induced by SEs.

**6. Therapy and prophylaxis** 

**6.1 Current therapeutic measures** 

**5.6 Nonhuman primate** 

As shown by studies in NHP, animals could be protected against SE intoxication using passive immunization with anti-SEB antibodies. The antibodies provided protection if given up to 4 hr after the NHPs had received 5 LD50sof aerosolized SEB (LeClaire et al., 2002). These studies showed that the antibodies were able to neutralize the toxin *in vivo* and provide protection after intoxication had occurred. Unfortunately, the antibody was a monoclonal antibody (Mab) created in a chicken, and the antibody itself may be antigenic and cause serum sickness. There are several anti-SEB Mab preparations that will not cause adverse reactions in humans. Because most Mabs are not developed using human cells, Mab development for human use will require "humanization of the Mab in order to eliminate the molecule's antigenicity (Goldsmith and Signore, 2010). Investigations using immune lymphocytes to prepare Mabs may provide therapeutics without the expense of humanization. Additionally, developing small-domain antibody fragments such as camilid antibodies may also provide therapeutic agents that, because of their size, may not be antigenic in humans (Graef et al., 2011). Further studies are needed to identify antibody reagents that will be successful therapeutics, and whether the antibody reagents will need to be combined with pharmacologic agents to enhance their efficacy.

#### **6.3 Possible pharmacologic agents to treat SE intoxication**

Over the past 20 years, there have been numerous investigations showing efficacy of various pharmacologic agents that prevent or delay lethality when animals are treated after a SE challenge, and as yet none has advanced to human clinical trials (Krakauer, 2010). Human activated protein C (hAPC) was approved for patients in severe septic shock, and had been used to treat TSS (DeVries et al. 2011). On October 25, 2011, Eli Lilly withdrew *Xigris (hAPC) because more in depth clinical trials indicated that drug did no better than a placebo in reducing mortality (*http://www.medscape.com/viewarticle/752169*).* The complexity of TSS tends to discount development of a single drug capable of treating the disease . Because the disease affects the endothelial cell vasculature of multiple organ systems, including drugs that treat endothelial cell damage should also help to establish a therapeutic regimen for TSS.

#### **6.4 Vaccines**

To protect soldiers against SEB exposure, the U. S. Army developed a formalin-treated SEB toxoid that had some degree of success in protecting animals against a SEB challenge (Tseng et al., 1995). Due to the fact that formalin treatment did not always inactivate every toxin molecule, there was a move to develop vaccines without the need for formalin

Staphylococcal Enterotoxins, Stayphylococcal Enterotoxin B and Bioterrorism 57

are easy to disseminate and produce moderate morbidity and low mortality. Category B agents do not meet criteria for use as a WMD, but dispersal of a Category B agent could

From all accounts, SEB meets the criteria for a Category B agent in that it is stable, easy to disseminate, and induces severe emesis and toxic shock. An aerosol of SEB in a crowded area could lead to an incapacitating disease in several hundred individuals. Although mortality would be low, the illness would create a serious public health impact by disrupting normal work days and cause havoc by increasing individual use of emergency

Many bioterrorism agents such as SEB are found in nature, are easy to isolate and produce in mass quantities and are usually stable in adverse environmental conditions (Ahanotu, et al., 2006). Because the agent is a common inhabitant in the environment, monitoring the agent becomes more difficult. The fact that there are accidental cases of food poisoning and occasional cases of TSS annually also complicates identifying bioterrorism incidents using SEB. In the final analysis, although SEB may not be the most favored bioterrorism agent, there is always a possibility that it will be used in an attack and, therefore, mechanisms

SEs are produced primarily by *S.aureus* which is a common inhabitant in the environment worldwide. SEs are a major cause of food poisoning and toxic shock syndrome. In the 1960s, SEB was weaponized as an incapacitating agent, and now is listed as a Category B bioterrorism agent. When inhaled, the toxin causes severe respiratory damage and endothelial dysfunction, often resulting in acute respiratory distress and severe lung damage. As yet, there is no FDA-approved vaccine or therapeutic agents to prevent or treat SEB-intoxication and with its ease of dissemination, SEB remains a serious bioterrorism

The work was supported by funds from the Defense Threat Reduction Agency (project CBM.THRTOX.03.10.RD.020). The opinions, interpretations, conclusions, and recommendations expressed in this publication are those of the author and are not

Abrahmsen, L., Dohlsten, M., Segren, S., Bjork, P., Johsson, E., and Kalland, T. (1995)

Acharya K. R., Baker M. D., (2004) Superantigen: structure-function relationships. Int. J.

Alber, G, Hammer, K., and Fleischer, B. (1990) Relationship between enterotoxic and T

staphylococcal enterotoxin A. EMBO J. 14: 2978-2986.

Characterization of two distinct MHC class II binding sites in the superantigen

lymphocyte-stimulating activity of staphylococcal enterotoxins. *J. Immunol*.

result in regional disruptions and hysteria.

should be in place for decontamination and treatment.

rooms (Ulrich et al., 1997).

**9. Summary** 

agent.

**10. Acknowledgements** 

**11. References** 

necessarily endorsed by the US Army.

Med. Microbiol. 293: 529-37.

144:4501-4506.

inactivation. A vaccine developed by site-specific mutagenesis provided safer and more effective vaccines. The most effective vaccine (SEBvax) was designed with mutations in the MHC class II binding region so that the vaccine no longer was capable of cross-linking T cells to APCs (Ulrich et al., 1998). The military is no longer funding development of SEBvax. The vaccine is now being used to develop a trivalent subunit vaccine that includes mutated Tst-1 and SEA proteins as well. The combination vaccine should provide protection against SEB but also against SEs more commonly associated with TSS (http://www.regionalinnovation.org/success.cfm?story=32).

### **7. Detection of SEs and SEB**

Since September 11, 2001, The U.S. and its allies have been concerned with detection of those agents that could be dispersed by aerosol (Kman and Bachman, 2011). There are several methods used for monitoring agents of bioterrorism and surveillance occurs through an umbrella of monitoring systems. A major component of surveillance, syndromic surveillance, results from the monitoring of clinical manifestations of certain illnesses to determine if there is a higher than normal number of cases. This is usually followed by laboratory surveillance in which certain markers and laboratory data indicate the presence of a bioterrorism agent. Another type of surveillance is environmental during which the environment is continually sampled for the presence of biological agents. In situations like SEB which is not generally monitored environmentally via the BioWatch Program, syndromic and laboratory surveillance becomes extremely important for monitoring bioterrorism attacks.

SEs are stable proteins and therefore identification of the proteins using anti-SEB reagents should be possible in both the field and medical facilities. Available immunoassays are capable of identifying the protein in picogram amounts and can be used to monitor samples taken from the environment (Kahn et al. 2003; Sapsford et al., 2005). Because most humans have been exposed to SEs and have developed antibodies against them, the presence of anti-SE antibodies is of little diagnostic value, but the detection of the toxin in body fluids or from nasal swabs (after an aerosol exposure), should provide a positive confirmation (Ulrich et al., 1997).

In cases of infection with *S. aureus*, polymerase chain reaction (PCR) assays can determine the presence of the SE gene (Chiang et al., 2008; Rajkovic et al., 2006). Particularly in the case of a toxin that is known to be present in staphylococcal infections, surveillance and monitoring at the clinical level is imperative for differentiating between random outbreaks of the disease and a bioterrorist attack.

#### **8. Category B biothreat agent**

Although redefined after September 11, 2001, rogue nations have used bioterrorism for centuries as a method to harm their opponents (Bellamy and Freedman, 2001; Phillips, 2005). As described by CDC, bioterrorism agents are separated into three categories for preparedness purposes depending upon their ease of dissemination, and the ability to cause excessive morbidity and mortality (Rotz et al, 2002). Category A includes agents such as Variola major (smallpox) and *Yersinia pestis* (plague) that have been used as a weapon of mass destruction (WMD) (Henderson, 1999). As previously mentioned, Category B agents are easy to disseminate and produce moderate morbidity and low mortality. Category B agents do not meet criteria for use as a WMD, but dispersal of a Category B agent could result in regional disruptions and hysteria.

From all accounts, SEB meets the criteria for a Category B agent in that it is stable, easy to disseminate, and induces severe emesis and toxic shock. An aerosol of SEB in a crowded area could lead to an incapacitating disease in several hundred individuals. Although mortality would be low, the illness would create a serious public health impact by disrupting normal work days and cause havoc by increasing individual use of emergency rooms (Ulrich et al., 1997).

Many bioterrorism agents such as SEB are found in nature, are easy to isolate and produce in mass quantities and are usually stable in adverse environmental conditions (Ahanotu, et al., 2006). Because the agent is a common inhabitant in the environment, monitoring the agent becomes more difficult. The fact that there are accidental cases of food poisoning and occasional cases of TSS annually also complicates identifying bioterrorism incidents using SEB. In the final analysis, although SEB may not be the most favored bioterrorism agent, there is always a possibility that it will be used in an attack and, therefore, mechanisms should be in place for decontamination and treatment.

#### **9. Summary**

56 Bioterrorism

inactivation. A vaccine developed by site-specific mutagenesis provided safer and more effective vaccines. The most effective vaccine (SEBvax) was designed with mutations in the MHC class II binding region so that the vaccine no longer was capable of cross-linking T cells to APCs (Ulrich et al., 1998). The military is no longer funding development of SEBvax. The vaccine is now being used to develop a trivalent subunit vaccine that includes mutated Tst-1 and SEA proteins as well. The combination vaccine should provide protection against SEB but also against SEs more commonly associated with TSS

Since September 11, 2001, The U.S. and its allies have been concerned with detection of those agents that could be dispersed by aerosol (Kman and Bachman, 2011). There are several methods used for monitoring agents of bioterrorism and surveillance occurs through an umbrella of monitoring systems. A major component of surveillance, syndromic surveillance, results from the monitoring of clinical manifestations of certain illnesses to determine if there is a higher than normal number of cases. This is usually followed by laboratory surveillance in which certain markers and laboratory data indicate the presence of a bioterrorism agent. Another type of surveillance is environmental during which the environment is continually sampled for the presence of biological agents. In situations like SEB which is not generally monitored environmentally via the BioWatch Program, syndromic and laboratory surveillance becomes extremely important for monitoring

SEs are stable proteins and therefore identification of the proteins using anti-SEB reagents should be possible in both the field and medical facilities. Available immunoassays are capable of identifying the protein in picogram amounts and can be used to monitor samples taken from the environment (Kahn et al. 2003; Sapsford et al., 2005). Because most humans have been exposed to SEs and have developed antibodies against them, the presence of anti-SE antibodies is of little diagnostic value, but the detection of the toxin in body fluids or from nasal swabs (after an aerosol exposure), should provide a positive confirmation (Ulrich

In cases of infection with *S. aureus*, polymerase chain reaction (PCR) assays can determine the presence of the SE gene (Chiang et al., 2008; Rajkovic et al., 2006). Particularly in the case of a toxin that is known to be present in staphylococcal infections, surveillance and monitoring at the clinical level is imperative for differentiating between random outbreaks

Although redefined after September 11, 2001, rogue nations have used bioterrorism for centuries as a method to harm their opponents (Bellamy and Freedman, 2001; Phillips, 2005). As described by CDC, bioterrorism agents are separated into three categories for preparedness purposes depending upon their ease of dissemination, and the ability to cause excessive morbidity and mortality (Rotz et al, 2002). Category A includes agents such as Variola major (smallpox) and *Yersinia pestis* (plague) that have been used as a weapon of mass destruction (WMD) (Henderson, 1999). As previously mentioned, Category B agents

(http://www.regionalinnovation.org/success.cfm?story=32).

**7. Detection of SEs and SEB** 

bioterrorism attacks.

et al., 1997).

of the disease and a bioterrorist attack.

**8. Category B biothreat agent** 

SEs are produced primarily by *S.aureus* which is a common inhabitant in the environment worldwide. SEs are a major cause of food poisoning and toxic shock syndrome. In the 1960s, SEB was weaponized as an incapacitating agent, and now is listed as a Category B bioterrorism agent. When inhaled, the toxin causes severe respiratory damage and endothelial dysfunction, often resulting in acute respiratory distress and severe lung damage. As yet, there is no FDA-approved vaccine or therapeutic agents to prevent or treat SEB-intoxication and with its ease of dissemination, SEB remains a serious bioterrorism agent.

#### **10. Acknowledgements**

The work was supported by funds from the Defense Threat Reduction Agency (project CBM.THRTOX.03.10.RD.020). The opinions, interpretations, conclusions, and recommendations expressed in this publication are those of the author and are not necessarily endorsed by the US Army.

### **11. References**


Staphylococcal Enterotoxins, Stayphylococcal Enterotoxin B and Bioterrorism 59

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**4** 

*Sweden* 

Rickard Knutsson

*National Veterinary Institute (SVA),* 

**Diagnostic Bioterrorism Response Strategies** 

Various biological agents such as bacteria, parasites, viruses and toxins may be deliberately released and spread through feed, food, water and air to cause harm and panic (Rotz et al., 2004). These biological agents can infect humans and animals but also crops (Gullino, 2008). Bioterrorism is probably the most inter-sectoral and international challenges among Chemical, Biological, Radiological, and Nuclear (CBRN) threats. To improve the interactions between these sectors there are some key issues involving R&D, training, event exercises, early warning and effective communication strategies that need to be addressed to rapidly share event information related to detection and identification. In this perspective, diagnostic capabilities are critical components to enhance the preparedness against bioterrorism (Morse, 2004). Covert and overt incidents will lead to various alarm chains. In a covert incident, which is characterized by an unannounced release, the early response and detection will be driven by public health organizations. However, an overt incident is characterized by the fact that the perpetrator announces responsibility and the response will therefore be driven by law enforcement. A diagnostic response strategy must be able to address both types of incidents. This requires a multidisciplinary network composed of diagnostic capabilities both in law enforcement agencies and public health organizations such as environmental, agricultural, veterinary, and food. As a result, laboratory response networks have been developed in different countries. The US Laboratory Response Network (LRN) was established in 1999. Its formation was based on a presidential order (Decision Directive 39) in which the Centers for Disease Control and Prevention (CDC), Association of Public Health Laboratories (APHL), Federal Bureau of Investigation (FBI), and United States Army Medical Research Institute of Infectious Diseases (USAMRIID) was involved (Morse, 2003). The objective of the US LRN is to ensure an effective laboratory response to bioterrorism by improving the law enforcement and public health laboratory infrastructure. The US LRN links local, state and national public health laboratories, as well as agriculture, veterinary, military, water- and food testing laboratories. In addition, the LRN links also to international laboratories in Canada, Australia, Japan, United Kingdom and Germany. Several other countries have developed similar laboratory networks such as Canada (CRTI, 2007), Australia (Editorial, 2004) and South Korea (Hwang, 2008). A Swedish LRN was established in 2009 with the aim of facilitating collaboration between law enforcement, first responders and public health agencies. The Swedish Forum for Biopreparedness Diagnostics (FBD) was established in 2007. FBD is a national laboratory multiagency cooperation, consisting of partners from the National Food Administration (SLV), the Swedish Defense Research Agency (FOI), the National Veterinary Institute (SVA) and the Swedish Institute

**1. Introduction** 


## **Diagnostic Bioterrorism Response Strategies**

#### Rickard Knutsson

*National Veterinary Institute (SVA), Sweden* 

#### **1. Introduction**

64 Bioterrorism

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beta-specific superantigen staphylococcal enterotoxin B: stimulation of mature T

P. M. (2002) Characterization and expression analysis of *Staphylococcus aureus* pathogenicity island3. Implications for the evolution of staphylococcal enterotoxin Various biological agents such as bacteria, parasites, viruses and toxins may be deliberately released and spread through feed, food, water and air to cause harm and panic (Rotz et al., 2004). These biological agents can infect humans and animals but also crops (Gullino, 2008). Bioterrorism is probably the most inter-sectoral and international challenges among Chemical, Biological, Radiological, and Nuclear (CBRN) threats. To improve the interactions between these sectors there are some key issues involving R&D, training, event exercises, early warning and effective communication strategies that need to be addressed to rapidly share event information related to detection and identification. In this perspective, diagnostic capabilities are critical components to enhance the preparedness against bioterrorism (Morse, 2004). Covert and overt incidents will lead to various alarm chains. In a covert incident, which is characterized by an unannounced release, the early response and detection will be driven by public health organizations. However, an overt incident is characterized by the fact that the perpetrator announces responsibility and the response will therefore be driven by law enforcement. A diagnostic response strategy must be able to address both types of incidents. This requires a multidisciplinary network composed of diagnostic capabilities both in law enforcement agencies and public health organizations such as environmental, agricultural, veterinary, and food. As a result, laboratory response networks have been developed in different countries. The US Laboratory Response Network (LRN) was established in 1999. Its formation was based on a presidential order (Decision Directive 39) in which the Centers for Disease Control and Prevention (CDC), Association of Public Health Laboratories (APHL), Federal Bureau of Investigation (FBI), and United States Army Medical Research Institute of Infectious Diseases (USAMRIID) was involved (Morse, 2003). The objective of the US LRN is to ensure an effective laboratory response to bioterrorism by improving the law enforcement and public health laboratory infrastructure. The US LRN links local, state and national public health laboratories, as well as agriculture, veterinary, military, water- and food testing laboratories. In addition, the LRN links also to international laboratories in Canada, Australia, Japan, United Kingdom and Germany. Several other countries have developed similar laboratory networks such as Canada (CRTI, 2007), Australia (Editorial, 2004) and South Korea (Hwang, 2008). A Swedish LRN was established in 2009 with the aim of facilitating collaboration between law enforcement, first responders and public health agencies. The Swedish Forum for Biopreparedness Diagnostics (FBD) was established in 2007. FBD is a national laboratory multiagency cooperation, consisting of partners from the National Food Administration (SLV), the Swedish Defense Research Agency (FOI), the National Veterinary Institute (SVA) and the Swedish Institute

Diagnostic Bioterrorism Response Strategies 67

The European political leadership has made efforts to improve a coordinated EU response to the bioterrorism incidents. (Sundelius et al., 2004) (Tegnell et al., 2003). The efforts have improved strategic, tactical, and operational aspects of preparedness planning and response (Brandeau et al., 2009). The strategic planning efforts have formed the basis for multisectoral R&D activities within the field of bioterrorism diagnostics and have improved the

Interagency and multi-sectoral laboratory cooperation requires a well developed infrastructure in terms of (i) communication and IT-systems, (ii) facilities, (iii) instruments/equipments and (iv) staff. A solid interagency cooperation of the public health laboratories, veterinary, agriculture, military, and water- and food-testing laboratories infrastructure must be based on strategic plans. These plans must facilitate building integrated response architectures and the promotion of coordination. Various tools, such as discrete event simulation modeling (Hupert et al., 2002) and information infrastructure tools (Kun et al., 2002) are useful in developing the interagency cooperation for laboratory

**Communication and IT-systems.** The laboratory infrastructure must include services to inform and communicate accurate diagnostic data at different levels (Zarcone et al., 2010). A key element for bioterrorism preparedness is information exchange and diseases outbreaks reporting (Horton et al., 2002). Surveillance systems are crucial for early warning of biothreat agents and a coordinated information infrastructure between surveillance and laboratory activities is needed. It has been found that there is a need for coordination between syndromic and laboratory based surveillance (Sintchenko et al., 2009). The diagnostic response strategies must simultaneously fit both the epidemiological and criminal investigations and ongoing activities to improve capabilities to share electronic laboratory diagnostic data (Zarcone et al., 2010). Rational communication procedures are a key mechanism to effective bioterrorism preparedness (Pien et al., 2006). Appropriate and secure communication tools are especially important in the alarm chain allowing police and first responders to contact public and animal health official in terms of covert and overt bioterrorism incident (Holmdahl et al,. 2011). In addition, it is important to facilitate communication between clinicians, sentinel laboratories and US LRN reference laboratories. A failure to communicate information may lead to delayed detection and a greater pressure to handle the incident (Pien et al., 2006). An important infrastructure feature is data handling and electronic information sharing. The US LRN includes approximately 1200 users which require a central point of contact (Morse, 2003). To securely share standard laboratory results between laboratories, LRN Results Messenger (LRN RM) has been established (CDC, 2007). The need for sharing data was clearly identified during the anthrax letter incident in 2001. Approximately 125,000 samples and more than 1 million tests were reported during the event (CDC, 2007). The lack of efficient data sharing tools made it difficult for the laboratories to share data. Laboratory results are critical during an outbreak and they facilitate the decision making. Therefore, to support early detection and response the laboratory should share data (CDC, 2007). The LRN RM has been installed in more than

4. Response and health alert network/communications and information technology;

5. Communicating Health Risks and Health Information Dissemination; and

6. Education and training (Kun et al., 2002).

bioterrorism preparedness.

laboratory response networks and interagency cooperation.

**3. Laboratory infrastructure and communication** 

for Communicable Disease Control (SMI). The aim of FBD is to strengthen the diagnostic capacity in Sweden regarding dangerous pathogens. The various laboratory networks that have been established in different countries have more or less the same objectives: rapid detection, identification and characterization of pathogens, targeted surveillance programs, strengthen laboratory response capacities and capabilities, and recovery. This includes; harmonization of diagnostic methods, increasing diagnostic capacity, training and exercises, interactions with other networks, and coordination of diagnostic emergency response. For these reasons, a broad diagnostic portfolio is needed in order to respond to covert and overt bioterrorism incidents. Diagnostic collaboration and networks are essential for an efficient response to a bioterrorism attack. Diagnostic response strategies must consider the abilities of the network in handling of the following: laboratories expertise, index case, decision making, tracing and tracking, and crime scene investigations.

#### **2. Strategic planning**

To obtain international multisectoral cooperation, in terms of bioterrorism prevention, policy makers have an important function. Policy makers at the local, regional, national and international levels must work in the same direction. However, this is not always realistic and as a result strategic planning is crucial, and the use of planning scenarios (DHS, 2005) can enhance strategic planning (Davis et al., 2007). Interagency collaborative efforts are one of the most critical factors to ensure an efficient bioterrorism preparedness and response plan. Several intergovernmental organizations such as INTERPOL (INTERPOL, 2010), World Health Organization (WHO), Food and Agricultural Organization (FAO) and World Organization for Animal Health (OIE) have ongoing programs and activities to counter the threat of attacks on humans, animals and plants (Pearson, 2006). The Biological and Toxin Weapon Convention (BTWC) prohibits the deliberate release of agents to attack plants, animals and humans (UN, 1972). However, effective prevention and countermeasures for deliberate attacks need to be developed in harmony with measures to control either natural or accidental outbreaks of disease.

A lot of strategic planning is taking place at the national level. For example, reports by the US Congressional Research Service identifies strategic planning as one of four critical areas of bioterrorism preparedness and that agency implementation will be a key component to translate strategic goals into effective programs and polices (Gottron, 2011). The European Union has also developed strategic plans on how to counter bioterrorism and CBRN attacks that are outlined in the EU CBRN Action Plan (EC, 2009). The Action Plan will be implemented in the period 2010 to 2014. Some examples of other countries focusing on strategic planning for bioterrorism are Canada (CRTI, 2007), South Korea (Hwang, 2008). A lot of progress has been made in strengthening local, regional/state, national and international capacities to detect and respond to bioterrorism since letters containing spores of *Bacillus anthracis* were sent via the US mail in 2001 (Rotz et al., 2004) (Smith, 2004). The anthrax letters caused the CDC to revise its strategic plan for bioterrorism preparedness and response (Koplan, 2001), which is focused on the following six focus areas:


for Communicable Disease Control (SMI). The aim of FBD is to strengthen the diagnostic capacity in Sweden regarding dangerous pathogens. The various laboratory networks that have been established in different countries have more or less the same objectives: rapid detection, identification and characterization of pathogens, targeted surveillance programs, strengthen laboratory response capacities and capabilities, and recovery. This includes; harmonization of diagnostic methods, increasing diagnostic capacity, training and exercises, interactions with other networks, and coordination of diagnostic emergency response. For these reasons, a broad diagnostic portfolio is needed in order to respond to covert and overt bioterrorism incidents. Diagnostic collaboration and networks are essential for an efficient response to a bioterrorism attack. Diagnostic response strategies must consider the abilities of the network in handling of the following: laboratories expertise, index case, decision

To obtain international multisectoral cooperation, in terms of bioterrorism prevention, policy makers have an important function. Policy makers at the local, regional, national and international levels must work in the same direction. However, this is not always realistic and as a result strategic planning is crucial, and the use of planning scenarios (DHS, 2005) can enhance strategic planning (Davis et al., 2007). Interagency collaborative efforts are one of the most critical factors to ensure an efficient bioterrorism preparedness and response plan. Several intergovernmental organizations such as INTERPOL (INTERPOL, 2010), World Health Organization (WHO), Food and Agricultural Organization (FAO) and World Organization for Animal Health (OIE) have ongoing programs and activities to counter the threat of attacks on humans, animals and plants (Pearson, 2006). The Biological and Toxin Weapon Convention (BTWC) prohibits the deliberate release of agents to attack plants, animals and humans (UN, 1972). However, effective prevention and countermeasures for deliberate attacks need to be developed in harmony with measures to control either natural

A lot of strategic planning is taking place at the national level. For example, reports by the US Congressional Research Service identifies strategic planning as one of four critical areas of bioterrorism preparedness and that agency implementation will be a key component to translate strategic goals into effective programs and polices (Gottron, 2011). The European Union has also developed strategic plans on how to counter bioterrorism and CBRN attacks that are outlined in the EU CBRN Action Plan (EC, 2009). The Action Plan will be implemented in the period 2010 to 2014. Some examples of other countries focusing on strategic planning for bioterrorism are Canada (CRTI, 2007), South Korea (Hwang, 2008). A lot of progress has been made in strengthening local, regional/state, national and international capacities to detect and respond to bioterrorism since letters containing spores of *Bacillus anthracis* were sent via the US mail in 2001 (Rotz et al., 2004) (Smith, 2004). The anthrax letters caused the CDC to revise its strategic plan for bioterrorism preparedness and

1. Preparedness planning and readiness assessment (including the National

3. Laboratory capacity including diagnosis and characterization of biological agents;

response (Koplan, 2001), which is focused on the following six focus areas:

making, tracing and tracking, and crime scene investigations.

**2. Strategic planning** 

or accidental outbreaks of disease.

Pharmaceutical Stockpile);

2. Detection, surveillance and epidemiology capacity;


The European political leadership has made efforts to improve a coordinated EU response to the bioterrorism incidents. (Sundelius et al., 2004) (Tegnell et al., 2003). The efforts have improved strategic, tactical, and operational aspects of preparedness planning and response (Brandeau et al., 2009). The strategic planning efforts have formed the basis for multisectoral R&D activities within the field of bioterrorism diagnostics and have improved the laboratory response networks and interagency cooperation.

#### **3. Laboratory infrastructure and communication**

Interagency and multi-sectoral laboratory cooperation requires a well developed infrastructure in terms of (i) communication and IT-systems, (ii) facilities, (iii) instruments/equipments and (iv) staff. A solid interagency cooperation of the public health laboratories, veterinary, agriculture, military, and water- and food-testing laboratories infrastructure must be based on strategic plans. These plans must facilitate building integrated response architectures and the promotion of coordination. Various tools, such as discrete event simulation modeling (Hupert et al., 2002) and information infrastructure tools (Kun et al., 2002) are useful in developing the interagency cooperation for laboratory bioterrorism preparedness.

**Communication and IT-systems.** The laboratory infrastructure must include services to inform and communicate accurate diagnostic data at different levels (Zarcone et al., 2010). A key element for bioterrorism preparedness is information exchange and diseases outbreaks reporting (Horton et al., 2002). Surveillance systems are crucial for early warning of biothreat agents and a coordinated information infrastructure between surveillance and laboratory activities is needed. It has been found that there is a need for coordination between syndromic and laboratory based surveillance (Sintchenko et al., 2009). The diagnostic response strategies must simultaneously fit both the epidemiological and criminal investigations and ongoing activities to improve capabilities to share electronic laboratory diagnostic data (Zarcone et al., 2010). Rational communication procedures are a key mechanism to effective bioterrorism preparedness (Pien et al., 2006). Appropriate and secure communication tools are especially important in the alarm chain allowing police and first responders to contact public and animal health official in terms of covert and overt bioterrorism incident (Holmdahl et al,. 2011). In addition, it is important to facilitate communication between clinicians, sentinel laboratories and US LRN reference laboratories. A failure to communicate information may lead to delayed detection and a greater pressure to handle the incident (Pien et al., 2006). An important infrastructure feature is data handling and electronic information sharing. The US LRN includes approximately 1200 users which require a central point of contact (Morse, 2003). To securely share standard laboratory results between laboratories, LRN Results Messenger (LRN RM) has been established (CDC, 2007). The need for sharing data was clearly identified during the anthrax letter incident in 2001. Approximately 125,000 samples and more than 1 million tests were reported during the event (CDC, 2007). The lack of efficient data sharing tools made it difficult for the laboratories to share data. Laboratory results are critical during an outbreak and they facilitate the decision making. Therefore, to support early detection and response the laboratory should share data (CDC, 2007). The LRN RM has been installed in more than

Diagnostic Bioterrorism Response Strategies 69

laboratories is based on traditional methods such as labor intensive cultivation, autopsy and necropsy. However, there is a need to have alternative methods for complementary tests such as molecular based instruments in a BSL-3 level or BSL-4 environment. It is therefore important to evaluate computers, DNA extraction robots and PCR equipment from an

Fig. 2. The unique potassium iodide KI-discus-test, to validate a class II safety cabinet with a DNA isolation robot in a BSL-3 laboratory. The KI discus test is defined in the European Standard for microbiological safety cabinets, EN12469:2000 as a test method for validating

**Staff.** Having trained personnel is critical for the overall bioterrorism preparedness effort (Barden, 2002) and leadership (Marshall et al., 2010). Additional training and education is necessary to work in BSL-3 and BSL-4 labs. Minimum requirements in terms of general biosafety training include definitions of biological and bio-hazardous materials, risk groups, biosafety containment levels, controls and protective clothing including staff at sentinel laboratories (Wagar et al., 2010). Various training courses are available and one example is a Bioterrorism Preparedness Training for LRN Sentinel Laboratories offered by the National Laboratory Training Network (NLTN, 2011). It is important to have training on various diagnostic methods as well as training related to biosafety and biosecurity (Kalish et al., 2009). Joint laboratory exercises are used to evaluate the laboratory organization. Education is also crucial in the other sectors such as veterinary laboratories

Many regulations, practices, programs and inspections have to be met and/or fulfilled to be allowed to work on biothreat agents. This requires inspections at organizational-, facility- and personnel level. The regulations and inspections differ from country to country and from sector to sector. The US Select Agents Program clearly regulates the use

the operator protection capabilities of the cabinet (Photo: SVA).

**4. Laboratory standards, certification and methods** 

(Lowenstine et al., 2006).

operational point of view for these laboratories.

150 LRN laboratories including public health, military, federal, food, veterinary and international labs (CDC, 2007). It allows storage and sharing of tests results for biological LRN assays. In addition, the data management supports electronic reporting of proficiency testing results and the ability of laboratories to review their test results and their performance.

**Facilities and laboratories.** To respond to human, animal and plant biothreat agents a number of laboratory facilities are needed; e.g. clinical laboratories, animal laboratories, plant laboratories, environmental laboratories, military laboratories and forensic laboratories. Most biothreat agents, which are also select agents (CDC) (APHIS), require various biosafety levels due to their pathogenic characteristics. According to work biosafety regulations agents such as *Variola major* (smallpox) and viral hemorrhagic fevers (Ebola, Marburg, etc) requires work at Biosafety Level 4 (BSL-4). Other agents such as *Bacillus anthracis*, *Fransicella tularensis*, and *Yersinia pestis* require BSL-3 laboratories. Foot and Mouth Disease virus (FMD), which is an animal pathogen, also requires a BSL-3 laboratory. In total, there are various laboratory levels such as BSL-2, BSL-3, BSL-4 and BSL-2 and BSL-3 animal facilities. All of these facilities have various design features and each facility has to fulfill different functional and operational goals.

Fig. 1. The US Laboratory Response Networks in terms of operational components; National laboratories to perform definitive characterization, Reference laboratories to perform confirmatory testing and Sentinel labs to recognize and rule-out (Nauschuetz, 2005).

**Instruments/equipments.** The laboratories must be equipped with appropriate and evaluated instruments that also are operational in a BSL-3 or BSL-4 laboratory. The interior of these labs must also be designed for different chemical and gas decontamination methods. Many BSL-3 and BSL-4 laboratories are composed of stainless steel to allow decontamination work of highly pathogenic microorganisms, which require the highest level of cleanliness and durability. Proper biosafety cabinets, autoclaves, glove boxes and ventilation systems must continuously be monitored and tested. A lot of work in these

150 LRN laboratories including public health, military, federal, food, veterinary and international labs (CDC, 2007). It allows storage and sharing of tests results for biological LRN assays. In addition, the data management supports electronic reporting of proficiency testing results and the ability of laboratories to review their test results and their

**Facilities and laboratories.** To respond to human, animal and plant biothreat agents a number of laboratory facilities are needed; e.g. clinical laboratories, animal laboratories, plant laboratories, environmental laboratories, military laboratories and forensic laboratories. Most biothreat agents, which are also select agents (CDC) (APHIS), require various biosafety levels due to their pathogenic characteristics. According to work biosafety regulations agents such as *Variola major* (smallpox) and viral hemorrhagic fevers (Ebola, Marburg, etc) requires work at Biosafety Level 4 (BSL-4). Other agents such as *Bacillus anthracis*, *Fransicella tularensis*, and *Yersinia pestis* require BSL-3 laboratories. Foot and Mouth Disease virus (FMD), which is an animal pathogen, also requires a BSL-3 laboratory. In total, there are various laboratory levels such as BSL-2, BSL-3, BSL-4 and BSL-2 and BSL-3 animal facilities. All of these facilities have various design features and each facility has to fulfill

Fig. 1. The US Laboratory Response Networks in terms of operational components; National laboratories to perform definitive characterization, Reference laboratories to perform confirmatory testing and Sentinel labs to recognize and rule-out (Nauschuetz, 2005).

**Instruments/equipments.** The laboratories must be equipped with appropriate and evaluated instruments that also are operational in a BSL-3 or BSL-4 laboratory. The interior of these labs must also be designed for different chemical and gas decontamination methods. Many BSL-3 and BSL-4 laboratories are composed of stainless steel to allow decontamination work of highly pathogenic microorganisms, which require the highest level of cleanliness and durability. Proper biosafety cabinets, autoclaves, glove boxes and ventilation systems must continuously be monitored and tested. A lot of work in these

performance.

different functional and operational goals.

laboratories is based on traditional methods such as labor intensive cultivation, autopsy and necropsy. However, there is a need to have alternative methods for complementary tests such as molecular based instruments in a BSL-3 level or BSL-4 environment. It is therefore important to evaluate computers, DNA extraction robots and PCR equipment from an operational point of view for these laboratories.

Fig. 2. The unique potassium iodide KI-discus-test, to validate a class II safety cabinet with a DNA isolation robot in a BSL-3 laboratory. The KI discus test is defined in the European Standard for microbiological safety cabinets, EN12469:2000 as a test method for validating the operator protection capabilities of the cabinet (Photo: SVA).

**Staff.** Having trained personnel is critical for the overall bioterrorism preparedness effort (Barden, 2002) and leadership (Marshall et al., 2010). Additional training and education is necessary to work in BSL-3 and BSL-4 labs. Minimum requirements in terms of general biosafety training include definitions of biological and bio-hazardous materials, risk groups, biosafety containment levels, controls and protective clothing including staff at sentinel laboratories (Wagar et al., 2010). Various training courses are available and one example is a Bioterrorism Preparedness Training for LRN Sentinel Laboratories offered by the National Laboratory Training Network (NLTN, 2011). It is important to have training on various diagnostic methods as well as training related to biosafety and biosecurity (Kalish et al., 2009). Joint laboratory exercises are used to evaluate the laboratory organization. Education is also crucial in the other sectors such as veterinary laboratories (Lowenstine et al., 2006).

#### **4. Laboratory standards, certification and methods**

Many regulations, practices, programs and inspections have to be met and/or fulfilled to be allowed to work on biothreat agents. This requires inspections at organizational-, facility- and personnel level. The regulations and inspections differ from country to country and from sector to sector. The US Select Agents Program clearly regulates the use

Diagnostic Bioterrorism Response Strategies 71

are developed in such a manner that they contain information for chain-of-custody

Law enforcement and public health must plan together to develop diagnostic response strategies for overt and covert bioterrorism incidents. This involves joint efforts to: (i) follow-up on lessons learned from previous incidents; (ii) scenario planning, training and exercises; (iii) R&D activities; and (iv) validate and implement methods in response plans. Methods for detection and identification of microorganisms and toxins forms the basis

**Lessons learned from bioterrorism incidents and biocrimes.** Several bioterrorism incidents and biocrimes have taken place, which have provided important lessons learned. For example, some more well known cases includes a salmonellosis outbreak in Oregon, a shigellosis outbreak in Dallas, the anthrax attacks of 2001 (amerithrax) and the the Aum Shinrikyo's attempt to develop biological weapons. The first case, the source of the 1984

> **Agent Distribution and**

*Salmonella typhimurium* ATCC 14028

*Bacillus anthracis* 

*Shigella dysenteriae* Type 2

*Bacillus anthracis* **transmission mode** 

bars/restaurants (blue cheese dressing, potato salad, lettuce)

Aerozolization of a liquid suspension of *B.* 

 Contamination of doughnuts and muffins

Letters with powder

*anthracis*

Sallad

**Reference** 

(Torok et al., 1997)

(Keim et al., 2001; Takahashi et al., 2004) (Olson, 1999)

(Kolavic et al., 1997)

(FBI, 2006) (Butler et al., 2002)

**location** 

1984, The Dalles, Oregon, USA

1993, Tokyo, Japan

1996, at a clinical laboratory in Dallas, Texas,

USA

2001, USA ("Amerithrax")

Table 1. Overview of some bioterrorism incidents/biocrimes (Dembek, 2007).

**5. Development of diagnostic response strategies** 

(Skinner et al., 2009) (Musshoff et al., 2009).

**Attackers Motive Year and** 

Religious motive to gain political control by influencing an election by making

An apocalyptic cult with a motive to trigger a world war

employee invited other laboratory workers to eat pastries in the coffee room

importance of his

research

voters ill.

Lone wolf A laboratory

Lone wolf Increase the

requirements.

Rajneeshee cult

Aum Shinrikyo

and transfer of specific biological agents and the program promotes laboratory safety and security (CDC, 2010). Other countries such as the United Kingdom, France, Denmark, Japan, Australia and Canada also have programs for governing facilities and personnel working on biothreat agents (NCBI, 2009). The Biological Weapon Convention (BTWC) (UN, 1972) and the United Nations Security Council (UNSCR1540, 2004) states that each nation should take action to implement national measures to avoid misuse of biological agents. However, other groups also exist. One example is the Australian Group, which is an informal forum of countries which seeks to ensure that exports do not contribute to the development of biological weapons (AG, 2010). These various programs and conventions to avoid misuse of biological agents shall be considered for laboratories working on biothreat agents.

**Laboratory standards.** In 2004 the World Health Organization (WHO) published the latest edition of their biosafety manual (WHO, 2004). After publication of this manual, WHO continued to work and in 2006 they published the *Biorisk Management: Laboratory Biosecurity Guidance* (WHO, 2006). This guide integrates biosafety and biosecurity. The European Committee for Standardization/Comité Européen de Normalisation (CEN) has continued to work on the WHO Biorisk management standard, and in 2008 CEN published Laboratory Biorisk Management Standard CWA 15793:2008 (CEN, 2008). The biorisk management standard provides guidance to an organization to identify, monitor and control laboratory biosafety and biosecurity in order to ensure that organizations are well prepared to respond if biological agents are released or go missing.

**Laboratory certification.** For laboratories working on biothreat, BSL-3 and BSL-4 agents, it is important to have a certified laboratory to confirm that the organization and facility is working with the highest and most appropriate standards. For this reason the organization must operate within international guidelines and national regulations. Facilities working on biothreat agents require laboratory or personnel certification (Gottron, 2011)*.* The laboratory certification process of BSL3- and BSL-4 laboratories involves compliance with a number of criteria in terms of bisafety and biosecurity. For instance, it includes a systematic review of safety processes within the laboratory such as personal protective equipment, building and system integrity and standard operating procedures (SOPs). It also includes administrative documentation and record retention systems. Therefore, to respond to a bioterrorism incident a laboratory certification process will form the basis for a well prepared and appropriate laboratory capability.

**Laboratory methods and harmonized protocols.** Harmonized methods and protocols are extremely important for multisectoral cooperation (Hodges et al., 2010). This means that standard protocols and reagents must be used to confirm tests. These activities can differ from country to country. To allow development of standardized and validated methods for detection and identification of biothreat agents, proficiency testing and ring trials by sending samples to different laboratories are required. For this reason a Critical Reagents Program (CRP) has been established in the US. The CRP collection includes inactivated antigens of select agent, genomic materials from biothreat agents and monoclonal and polyclonal antibodies of biothreat agents (CRP, 2011). This is a key component of laboratory preparedness as shortages of critical reagents will significantly influence the response testing (APHL, 2006). Based on validated reagents new methods can be developed, evaluated and tested (Donoso Mantke et al., 2005). The LRN laboratories use standardized protocols and reagents to detect and identify biothreat agents. The standardized protocols

and transfer of specific biological agents and the program promotes laboratory safety and security (CDC, 2010). Other countries such as the United Kingdom, France, Denmark, Japan, Australia and Canada also have programs for governing facilities and personnel working on biothreat agents (NCBI, 2009). The Biological Weapon Convention (BTWC) (UN, 1972) and the United Nations Security Council (UNSCR1540, 2004) states that each nation should take action to implement national measures to avoid misuse of biological agents. However, other groups also exist. One example is the Australian Group, which is an informal forum of countries which seeks to ensure that exports do not contribute to the development of biological weapons (AG, 2010). These various programs and conventions to avoid misuse of biological agents shall be considered for laboratories working on

**Laboratory standards.** In 2004 the World Health Organization (WHO) published the latest edition of their biosafety manual (WHO, 2004). After publication of this manual, WHO continued to work and in 2006 they published the *Biorisk Management: Laboratory Biosecurity Guidance* (WHO, 2006). This guide integrates biosafety and biosecurity. The European Committee for Standardization/Comité Européen de Normalisation (CEN) has continued to work on the WHO Biorisk management standard, and in 2008 CEN published Laboratory Biorisk Management Standard CWA 15793:2008 (CEN, 2008). The biorisk management standard provides guidance to an organization to identify, monitor and control laboratory biosafety and biosecurity in order to ensure that organizations are well prepared to respond

**Laboratory certification.** For laboratories working on biothreat, BSL-3 and BSL-4 agents, it is important to have a certified laboratory to confirm that the organization and facility is working with the highest and most appropriate standards. For this reason the organization must operate within international guidelines and national regulations. Facilities working on biothreat agents require laboratory or personnel certification (Gottron, 2011)*.* The laboratory certification process of BSL3- and BSL-4 laboratories involves compliance with a number of criteria in terms of bisafety and biosecurity. For instance, it includes a systematic review of safety processes within the laboratory such as personal protective equipment, building and system integrity and standard operating procedures (SOPs). It also includes administrative documentation and record retention systems. Therefore, to respond to a bioterrorism incident a laboratory certification process will form the basis for a well prepared and

**Laboratory methods and harmonized protocols.** Harmonized methods and protocols are extremely important for multisectoral cooperation (Hodges et al., 2010). This means that standard protocols and reagents must be used to confirm tests. These activities can differ from country to country. To allow development of standardized and validated methods for detection and identification of biothreat agents, proficiency testing and ring trials by sending samples to different laboratories are required. For this reason a Critical Reagents Program (CRP) has been established in the US. The CRP collection includes inactivated antigens of select agent, genomic materials from biothreat agents and monoclonal and polyclonal antibodies of biothreat agents (CRP, 2011). This is a key component of laboratory preparedness as shortages of critical reagents will significantly influence the response testing (APHL, 2006). Based on validated reagents new methods can be developed, evaluated and tested (Donoso Mantke et al., 2005). The LRN laboratories use standardized protocols and reagents to detect and identify biothreat agents. The standardized protocols

biothreat agents.

if biological agents are released or go missing.

appropriate laboratory capability.

are developed in such a manner that they contain information for chain-of-custody requirements.

#### **5. Development of diagnostic response strategies**

Law enforcement and public health must plan together to develop diagnostic response strategies for overt and covert bioterrorism incidents. This involves joint efforts to: (i) follow-up on lessons learned from previous incidents; (ii) scenario planning, training and exercises; (iii) R&D activities; and (iv) validate and implement methods in response plans. Methods for detection and identification of microorganisms and toxins forms the basis (Skinner et al., 2009) (Musshoff et al., 2009).

**Lessons learned from bioterrorism incidents and biocrimes.** Several bioterrorism incidents and biocrimes have taken place, which have provided important lessons learned. For example, some more well known cases includes a salmonellosis outbreak in Oregon, a shigellosis outbreak in Dallas, the anthrax attacks of 2001 (amerithrax) and the the Aum Shinrikyo's attempt to develop biological weapons. The first case, the source of the 1984


Table 1. Overview of some bioterrorism incidents/biocrimes (Dembek, 2007).

Diagnostic Bioterrorism Response Strategies 73

were infected with *B. anthracis*. Before the end of 2001, 22 cases of anthrax and 5 deaths had been reported. All of the anthrax cases were among postal workers or persons who had been in contact with contaminated mail. Exceptional collaboration was required from the different agencies involved. This event emphasized the importance of conducting public health and criminal investigations at the same time. The LRN served as a resource for identifying the agent in both environmental and clinical samples. An important lesson learned from this outbreak is that fine particles of a biological agent can become airborne, thus contaminating areas and placing persons at a risk and the need of microbial forensic

There are many lessons to be learned from these incidents. Different types of attackers such as extremist groups and lone wolves have been involved. In addition, these incidents necessitate that forensic and epidemiological investigations occur at the same time. For example, to provide the link between isolates from clinical and various environmental and food samples with the person responsible for the deliberate spread. The need for multiple

**Preparedness scenarios, training and exercises:** The use of planning and preparedness scenarios between public health and law enforcement organizations will contribute to developing diagnostic response strategies. Different software tools are available for different preparedness applications. Over the last 10 years, various scenarios have been developed and used for different purposes. Modeling and bioterrorism scenarios have been used to evaluate responses to attacks with different agents causing diseases such as anthrax (Zaric et al., 2008), (Hupert et al., 2009), Foot and Mouth Disease (Schoenbaum et al., 2003), and Qfever (Pappas et al., 2007), as well as decision making (O'Toole et al., 2001) and Bayesian approaches for estimating bioterror attacks (Ray et al., 2011). Different scenarios and exercises have been used to improve various counter measures such as a local bioterrorism exercise (Hoffman et al., 2000), an exercise on threat assessment and quantitative risk assessment (Zilinskas et al., 2004), and an exercise training bioterrorism surveillance system (Berndt, 2003). Because there are a number of published models and scenarios available these can be used to develop and improve scenarios to challenge diagnostic response strategies in terms of coordination, capabilities and capacity. Results and output from these

response teams has also been identified as a lesson learned.

scenarios, training and exercises can be used to initiate new R&D activities.

**R&D activities.** Joint R&D activities between first responders, forensic institutes and public health officials will contribute to developing appropriate methods. However this requires strategic planning and a laboratory infrastructure. Many R&D activities are performed in a specific sector, such as public health, animal health, food safety and law enforcement. Over the last few years joint diagnostic methods have been developed to counter bioterrorism. However, a lot of research has been performed without questioning the different diagnostic end-users at local, regional or national level and lessons learned from incidents and exercises have not always been considered. R&D activities have involved a broad spectra of methods such as electron microscopy (Goldsmith et al., 2009), new molecular methods (Casman, 2004), automated testing (Byrne et al., 2003) and screening (Emanuel et al., 2005), immunoassays for toxins (2008), microarray and multiplexing and nanotechnology methods (Menezes, 2011). Although technology has improved significantly since 2001 many diagnostic methods are still based on immunoassays, ELISA and PCR (Kellogg, 2010). First responders uses primarily field

(Dance, 2006).

salmonellosis outbreak in The Dalles, Oregon, was puzzling beacuse the epidemiological investigation revealed multiple items of food were involved instead of a single suspect item (Table 1). In total, 751 salmonellosis cases were identified and 45 persons were hospitalized.

This was a deliberate outbreak perpetrated by members of the Rajneeshee cult. The cult legally obtained *Salmonella* Typhimurium ATCC 14028 and spread cultures of this organism on salad bars in area restaurants. The cause of the outbreak was found to be due to intentional contamination in October 1985, when the Federal Bureau of Investigation (FBI) investigated the cult (McDade et al., 1998)**.** The FBI together with an Oregon public health laboratory official found an open vial of the strain in their laboratory more than a year after the outbreak took place. The culture of *Salmonella* Typhimurium ATCC 14028 found at the Rajneeshees farm was identical and indistinguishable from the outbreak strain that was isolated from clinical specimens and food items. The retrospective epidemiology was consistent and the deliberate contamination of the salad bars was confirmed (Torok et al., 1997). The case demonstrated the need for having joint cooperation between law enforcement and public health investigations. In addition, the case showed that many different food items and matrices may be responsible for a deliberate foodborne outbreak challenging the diagnostic capabilities. Various sample preparations methods for the different food items and food matrices will be needed.

In 1993 the Japanese Aum Shinrikyo cult released aerosolized spores of *B. anthracis* on two occasions. The first event took place in June when the cult sprayed *B. anthracis* from the roof of a building in downtown Tokyo. A month later the cult sprayed *B. anthracis* from a moving truck onto and around the Imperial Place and the Japan's parliament building in Tokyo (Dembek, 2007). However, none of the attacks led to any anthrax cases. In 2001, samples collected from the exterior of the exposed buildings in Tokyo were analyzed and it was found that the *B. anthracis* isolates were similar to the Sterne 34F2 strain, which is the strain used in animal vaccines for anthrax and is regarded as nonpathogenic for immunocompetent individuals. The release of this strain had little possibility of causing harm or death (Takahashi et al., 2004). From this incident one can learn that environmental sampling and proper storage is important. It also showed that microbial forensics is important as it enabled the investigators to identify the strain of *B. anthracis* released 8 years after the incident (Keim et al., 2001).

During the period between October 29th and November 1, 1996, 13 workers at a clinical laboratory in Dallas, Texas developed acute and severe diarrhea after consumption of muffins or doughnuts (Carus, 2001). The pathogen *Shigella dysenteriae* type 2 was isolated from stool samples from the infected workers. This pathogen is uncommon and no other shigellosis outbreaks occurred in the US at that time. Furthermore, no work on *Shigella* had taken place at that clinical laboratory. However, an examination of the freezer in the clinical laboratory showed some evidence of tampering with reference cultures of *S. dysenteriae* type 2. In August 1997, a laboratory technician was convicted of deliberately infecting coworkers with *Shigella dysenteriae* type 2 and sentenced to 20 years in prison (Everett, 2002). The laboratory and epidemiological investigations revealed a match of the laboratory strain to those isolated from food and clinical specimens. The tracing and epidemiological study was helped by the fact that only postproduction adulteration of the baked muffins and doughnuts could have resulted in their successful contamination.

On October 4, 2001, shortly after 9/11, an inhalation anthrax case was reported in a 63-old male in Florida (Fennelly et al., 2004)**.** Subsequently, additional persons were identified who

salmonellosis outbreak in The Dalles, Oregon, was puzzling beacuse the epidemiological investigation revealed multiple items of food were involved instead of a single suspect item (Table 1). In total, 751 salmonellosis cases were identified and 45 persons were hospitalized. This was a deliberate outbreak perpetrated by members of the Rajneeshee cult. The cult legally obtained *Salmonella* Typhimurium ATCC 14028 and spread cultures of this organism on salad bars in area restaurants. The cause of the outbreak was found to be due to intentional contamination in October 1985, when the Federal Bureau of Investigation (FBI) investigated the cult (McDade et al., 1998)**.** The FBI together with an Oregon public health laboratory official found an open vial of the strain in their laboratory more than a year after the outbreak took place. The culture of *Salmonella* Typhimurium ATCC 14028 found at the Rajneeshees farm was identical and indistinguishable from the outbreak strain that was isolated from clinical specimens and food items. The retrospective epidemiology was consistent and the deliberate contamination of the salad bars was confirmed (Torok et al., 1997). The case demonstrated the need for having joint cooperation between law enforcement and public health investigations. In addition, the case showed that many different food items and matrices may be responsible for a deliberate foodborne outbreak challenging the diagnostic capabilities. Various sample preparations methods for the

In 1993 the Japanese Aum Shinrikyo cult released aerosolized spores of *B. anthracis* on two occasions. The first event took place in June when the cult sprayed *B. anthracis* from the roof of a building in downtown Tokyo. A month later the cult sprayed *B. anthracis* from a moving truck onto and around the Imperial Place and the Japan's parliament building in Tokyo (Dembek, 2007). However, none of the attacks led to any anthrax cases. In 2001, samples collected from the exterior of the exposed buildings in Tokyo were analyzed and it was found that the *B. anthracis* isolates were similar to the Sterne 34F2 strain, which is the strain used in animal vaccines for anthrax and is regarded as nonpathogenic for immunocompetent individuals. The release of this strain had little possibility of causing harm or death (Takahashi et al., 2004). From this incident one can learn that environmental sampling and proper storage is important. It also showed that microbial forensics is important as it enabled the investigators to identify the strain of *B. anthracis* released 8 years

During the period between October 29th and November 1, 1996, 13 workers at a clinical laboratory in Dallas, Texas developed acute and severe diarrhea after consumption of muffins or doughnuts (Carus, 2001). The pathogen *Shigella dysenteriae* type 2 was isolated from stool samples from the infected workers. This pathogen is uncommon and no other shigellosis outbreaks occurred in the US at that time. Furthermore, no work on *Shigella* had taken place at that clinical laboratory. However, an examination of the freezer in the clinical laboratory showed some evidence of tampering with reference cultures of *S. dysenteriae* type 2. In August 1997, a laboratory technician was convicted of deliberately infecting coworkers with *Shigella dysenteriae* type 2 and sentenced to 20 years in prison (Everett, 2002). The laboratory and epidemiological investigations revealed a match of the laboratory strain to those isolated from food and clinical specimens. The tracing and epidemiological study was helped by the fact that only postproduction adulteration of the baked muffins and

On October 4, 2001, shortly after 9/11, an inhalation anthrax case was reported in a 63-old male in Florida (Fennelly et al., 2004)**.** Subsequently, additional persons were identified who

doughnuts could have resulted in their successful contamination.

different food items and food matrices will be needed.

after the incident (Keim et al., 2001).

were infected with *B. anthracis*. Before the end of 2001, 22 cases of anthrax and 5 deaths had been reported. All of the anthrax cases were among postal workers or persons who had been in contact with contaminated mail. Exceptional collaboration was required from the different agencies involved. This event emphasized the importance of conducting public health and criminal investigations at the same time. The LRN served as a resource for identifying the agent in both environmental and clinical samples. An important lesson learned from this outbreak is that fine particles of a biological agent can become airborne, thus contaminating areas and placing persons at a risk and the need of microbial forensic (Dance, 2006).

There are many lessons to be learned from these incidents. Different types of attackers such as extremist groups and lone wolves have been involved. In addition, these incidents necessitate that forensic and epidemiological investigations occur at the same time. For example, to provide the link between isolates from clinical and various environmental and food samples with the person responsible for the deliberate spread. The need for multiple response teams has also been identified as a lesson learned.

**Preparedness scenarios, training and exercises:** The use of planning and preparedness scenarios between public health and law enforcement organizations will contribute to developing diagnostic response strategies. Different software tools are available for different preparedness applications. Over the last 10 years, various scenarios have been developed and used for different purposes. Modeling and bioterrorism scenarios have been used to evaluate responses to attacks with different agents causing diseases such as anthrax (Zaric et al., 2008), (Hupert et al., 2009), Foot and Mouth Disease (Schoenbaum et al., 2003), and Qfever (Pappas et al., 2007), as well as decision making (O'Toole et al., 2001) and Bayesian approaches for estimating bioterror attacks (Ray et al., 2011). Different scenarios and exercises have been used to improve various counter measures such as a local bioterrorism exercise (Hoffman et al., 2000), an exercise on threat assessment and quantitative risk assessment (Zilinskas et al., 2004), and an exercise training bioterrorism surveillance system (Berndt, 2003). Because there are a number of published models and scenarios available these can be used to develop and improve scenarios to challenge diagnostic response strategies in terms of coordination, capabilities and capacity. Results and output from these scenarios, training and exercises can be used to initiate new R&D activities.

**R&D activities.** Joint R&D activities between first responders, forensic institutes and public health officials will contribute to developing appropriate methods. However this requires strategic planning and a laboratory infrastructure. Many R&D activities are performed in a specific sector, such as public health, animal health, food safety and law enforcement. Over the last few years joint diagnostic methods have been developed to counter bioterrorism. However, a lot of research has been performed without questioning the different diagnostic end-users at local, regional or national level and lessons learned from incidents and exercises have not always been considered. R&D activities have involved a broad spectra of methods such as electron microscopy (Goldsmith et al., 2009), new molecular methods (Casman, 2004), automated testing (Byrne et al., 2003) and screening (Emanuel et al., 2005), immunoassays for toxins (2008), microarray and multiplexing and nanotechnology methods (Menezes, 2011). Although technology has improved significantly since 2001 many diagnostic methods are still based on immunoassays, ELISA and PCR (Kellogg, 2010). First responders uses primarily field

Diagnostic Bioterrorism Response Strategies 75

of multidisciplinary detection technologies related to sampling, sample preparation, biomarker discovery, multiplexing and high resolution diagnostic typing tools (Knutsson et al., 2011). Clear mandate for coordination of response mechanisms is crucial and strong links between early warning systems provide a basis for the diagnostic bioterrorism

**Coordination – public health and forensic laboratories.** Full international cooperation and efficient detection technologies are essential in order to respond efficiently to a bioterrorism event. Considering the detection needs for covert and overt bioterrorism events will require a broad range of analytical tools. There are many promising technologies on the market but still there is a need to develop emerging technologies for different end-users. This can be promoted by multidisciplinary cooperation between first responders, forensic institutes and diagnostic laboratories representing LRNs. First responders have prerequisites to use the technology for a rapid identification of the agent on site and at the crime scene. The detection technology must be user friendly and allow usage in hot, warm and cold emergency zones. Forensic institutes' major interest is to maintain chain of custody and have methods that are validated for use in court, including methods for evaluation of the results given the circumstances of the case (forensic interpretation). Public and animal health diagnostic laboratories have in general other needs. They must have a broad range of diagnostic methods available for further characterization and typing of the etiological agent. In general, joint response teams and the coordination and back-up of different laboratories are therefore crucial. The decision making procedure is very important and it has been described that in response to bioterrorism clinicians must make decisions in 4 critical domains (diagnosis, management, prevention and reporting to public health) and public health organizations must make decisions in 4 other domains (interpretation of bioterrorism surveillance data, outbreak investigation, outbreak control and communication) (Bravata et al., 2004). Early warning and coordinated rapid detection is a backbone and therefore the physicians' ability to recognize potential cases in the identification and treatment of diseases associated with bioterrorism is crucial (Bush et al, 2001), as well as for veterinarians (Davis, 2004). Bioterrorism response clinicians are essential partners to LRNs (Gerberding et al., 2002) and especially at sentinel laboratories (Pien et al., 2006). Examples of the importance of the clinicians work is clearly documented (Maillard et al., 2002) (Harris et al., 2011) and also

the consequences if the diseases is not identified (Harris et al., 2011).

in different countries and in different types of laboratories.

**Capability and harmonization.** To announce, for instance an anthrax outbreak (Sternberg Lewerin et al., 2010), and to make a declaration of an incident, decision makers needs validated methods. Standardized and validated PCR assays for high risk agents, such as *B. anthracis* (Wielinga et al., 2011) (Scarlata et al., 2010) and *C. botulinum neurotoxin* (Fenicia et al., 2011) are fundamental for confirming disease outbreaks. The methods should be tested

**Capacity and robustness.** Initial sampling and rapid detection is crucial (Leport et al., 2011). However, feed, food, environmental and clinical samples all contains components that may inhibit the analysis (e.g., PCR-inhibitors). Appropriate sampling (Knutsson et al., 2003) and pre PCR processing strategies are therefore needed in order to circumvent inhibition. For this purpose models to investigate PCR inhibition is an important step to study and evaluate prior to applying a method for a specific purpose (Knutsson et al., 2002) (Knutsson et al, 2002). Another important function is to have a laboratory surge capability in different areas in order to improve the robustness of the diagnostic capacity during a bioterrorism incident.

response strategies.

based immunoassays and portable PCR-assays for biothreat detection, and local and sentinel public health laboratories uses traditional culture and biochemical assays, ELISAs and molecular-based PCR methods for biothreat identification. A problem with conventional detection methods are the lack of positive controls since these methods are based on living organisms. Rapid detection methods have been well investigated (Canton, 2005) (Peruski et al., 2003). The most useful technology for identification of biothreat agents is real-time PCR. However, microarray and multiplex assays for detection of biothreat agents, such as *Bacillus anthracis*, *Francisella tularensis* and *Yersinia pestis* by using multiplex qPCR have recently improved (Janse et al., 2010).

**Implementation in response plans.** Once new diagnostic methods are developed, they need to be evaluated and validated. This is an important step and requires access to reagents for proficiency testing and ring trial evaluations. Methods need to be validated for use in real incidents. The methods must also full fill requirements for forensic applications, which adds another aspect, see Table 2.


Table 2. A Bioterrorism response matrix outlining laboratory support and the multidisciplinary cooperation.

#### **6. Challenges – coordination, capability and capacity**

Diagnostic bioterrorism response strategies shall consider coordination/resilience, harmonization, robustness and high- resolution diagnostic tools. This includes R&D efforts

based immunoassays and portable PCR-assays for biothreat detection, and local and sentinel public health laboratories uses traditional culture and biochemical assays, ELISAs and molecular-based PCR methods for biothreat identification. A problem with conventional detection methods are the lack of positive controls since these methods are based on living organisms. Rapid detection methods have been well investigated (Canton, 2005) (Peruski et al., 2003). The most useful technology for identification of biothreat agents is real-time PCR. However, microarray and multiplex assays for detection of biothreat agents, such as *Bacillus anthracis*, *Francisella tularensis* and *Yersinia pestis* by using

**Implementation in response plans.** Once new diagnostic methods are developed, they need to be evaluated and validated. This is an important step and requires access to reagents for proficiency testing and ring trial evaluations. Methods need to be validated for use in real incidents. The methods must also full fill requirements for forensic applications, which adds

> Veterinarians/ First responders

Food inspectors/ First responders

Environmental inspectors/ First responders

Agricultural inspectors/ First responders

First responders

Diagnostic bioterrorism response strategies shall consider coordination/resilience, harmonization, robustness and high- resolution diagnostic tools. This includes R&D efforts

Physicians/ First responders **Laboratory Response** 

Veterinary laboratories

Water and food laboratories

Environmental laboratories

Agricultural and veterinary Laboratories

Clinical laboratories (local, regional, national)

laboratories

Food laboratories Forensic

**Forensics** 

Forensic laboratories

Forensic laboratories

Forensic laboratories

Forensic laboratories

laboratories

Forensic laboratories

Forensic laboratories

**Alarm (covert and/overt)** 

multiplex qPCR have recently improved (Janse et al., 2010).

another aspect, see Table 2.

**Sample Monitoring and** 

**Animal** Animal Health

**Environmental** Environmental

**Feed** Agricultural,

**Human clinical** Human

multidisciplinary cooperation.

**Drinking water** Food and

**surveillance** 

and Animal surveillance

Environmental monitoring

Food and Animal

**Food** Food surveillance Food inspectors/

**Plant** Plant surveillance Plant inspectors Agricultural

**6. Challenges – coordination, capability and capacity** 

Table 2. A Bioterrorism response matrix outlining laboratory support and the

monitoring

Health

syndromic surveillance of multidisciplinary detection technologies related to sampling, sample preparation, biomarker discovery, multiplexing and high resolution diagnostic typing tools (Knutsson et al., 2011). Clear mandate for coordination of response mechanisms is crucial and strong links between early warning systems provide a basis for the diagnostic bioterrorism response strategies.

**Coordination – public health and forensic laboratories.** Full international cooperation and efficient detection technologies are essential in order to respond efficiently to a bioterrorism event. Considering the detection needs for covert and overt bioterrorism events will require a broad range of analytical tools. There are many promising technologies on the market but still there is a need to develop emerging technologies for different end-users. This can be promoted by multidisciplinary cooperation between first responders, forensic institutes and diagnostic laboratories representing LRNs. First responders have prerequisites to use the technology for a rapid identification of the agent on site and at the crime scene. The detection technology must be user friendly and allow usage in hot, warm and cold emergency zones. Forensic institutes' major interest is to maintain chain of custody and have methods that are validated for use in court, including methods for evaluation of the results given the circumstances of the case (forensic interpretation). Public and animal health diagnostic laboratories have in general other needs. They must have a broad range of diagnostic methods available for further characterization and typing of the etiological agent. In general, joint response teams and the coordination and back-up of different laboratories are therefore crucial. The decision making procedure is very important and it has been described that in response to bioterrorism clinicians must make decisions in 4 critical domains (diagnosis, management, prevention and reporting to public health) and public health organizations must make decisions in 4 other domains (interpretation of bioterrorism surveillance data, outbreak investigation, outbreak control and communication) (Bravata et al., 2004). Early warning and coordinated rapid detection is a backbone and therefore the physicians' ability to recognize potential cases in the identification and treatment of diseases associated with bioterrorism is crucial (Bush et al, 2001), as well as for veterinarians (Davis, 2004). Bioterrorism response clinicians are essential partners to LRNs (Gerberding et al., 2002) and especially at sentinel laboratories (Pien et al., 2006). Examples of the importance of the clinicians work is clearly documented (Maillard et al., 2002) (Harris et al., 2011) and also the consequences if the diseases is not identified (Harris et al., 2011).

**Capability and harmonization.** To announce, for instance an anthrax outbreak (Sternberg Lewerin et al., 2010), and to make a declaration of an incident, decision makers needs validated methods. Standardized and validated PCR assays for high risk agents, such as *B. anthracis* (Wielinga et al., 2011) (Scarlata et al., 2010) and *C. botulinum neurotoxin* (Fenicia et al., 2011) are fundamental for confirming disease outbreaks. The methods should be tested in different countries and in different types of laboratories.

**Capacity and robustness.** Initial sampling and rapid detection is crucial (Leport et al., 2011). However, feed, food, environmental and clinical samples all contains components that may inhibit the analysis (e.g., PCR-inhibitors). Appropriate sampling (Knutsson et al., 2003) and pre PCR processing strategies are therefore needed in order to circumvent inhibition. For this purpose models to investigate PCR inhibition is an important step to study and evaluate prior to applying a method for a specific purpose (Knutsson et al., 2002) (Knutsson et al, 2002). Another important function is to have a laboratory surge capability in different areas in order to improve the robustness of the diagnostic capacity during a bioterrorism incident.

Diagnostic Bioterrorism Response Strategies 77

Home/2009/ISEC/AG/191) with the financial support from the Prevention of and Fight against Crime Programme of the European Union, European Commission – Directorate General Home Affairs. This publication reflects the views of the author, and the European Commission cannot be held responsible for any use which may be made of the information contained therein. A special thanks to Communication Officer Jeffrey Skiby (DTU-Food, Denmark) for critical reading and suggestion and to Professor Birgitta Rasmusson (the Swedish National Laboratory of Forensic Science, SKL) for critical reading with a focus on forensics and Dr Gary Barker (Institute of Food Research, IFR, United Kingdom) for

valuable comments on the section of diagnostic response strategies.

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Fig. 3. Up-scaling capabilities by the use of automated DNA extractions robot in a BSL-3 laboratory (Photo: SVA).

**High-resolution diagnostic tools.** Multiplexing strategies for detection of several biomarkers (Lindberg et al., 2010), as well as various molecular typing methods are useful for crime scene investigation, but also for tracing and tracking the deliberate contamination. The use of massive parallel sequencing will be useful to study strain isolates from a suspicious deliberate contamination event. By applying bioinformatics it is possible to rapidly analyze large amounts of sequence data with minimal post-processing time (Segerman et al., 2011).

#### **7. Conclusions**

To obtain diagnostic bioterrorism response strategies a number of issues must be solved including strategic planning, laboratory infrastructure and standards. The laboratory work has to be strongly linked to different early warning and surveillance systems. Multiple teams as well as joint laboratory protocols are also important resources to have in place. The development of diagnostic bioterrorism response strategies should be based on lessons learned from previous attacks/incidents, planning scenarios, R&D activities and validation and implementations of methods. Laboratories must be able to detect, identify, respond and recover from covert and overt bioterrorism incidents. Key recommendations include:


#### **8. Acknowledgements**

Writing of this chapter has been supported by grants from the Swedish Civil Contingencies Agency (Anslag 2:4 Krisberedskap), Sweden (Swedish Laboratory Response Network), and the framework of the EU-project AniBioThreat (Grant Agreement: Home/2009/ISEC/AG/191) with the financial support from the Prevention of and Fight against Crime Programme of the European Union, European Commission – Directorate General Home Affairs. This publication reflects the views of the author, and the European Commission cannot be held responsible for any use which may be made of the information contained therein. A special thanks to Communication Officer Jeffrey Skiby (DTU-Food, Denmark) for critical reading and suggestion and to Professor Birgitta Rasmusson (the Swedish National Laboratory of Forensic Science, SKL) for critical reading with a focus on forensics and Dr Gary Barker (Institute of Food Research, IFR, United Kingdom) for valuable comments on the section of diagnostic response strategies.

#### **9. References**

76 Bioterrorism

Fig. 3. Up-scaling capabilities by the use of automated DNA extractions robot in a BSL-3

**High-resolution diagnostic tools.** Multiplexing strategies for detection of several biomarkers (Lindberg et al., 2010), as well as various molecular typing methods are useful for crime scene investigation, but also for tracing and tracking the deliberate contamination. The use of massive parallel sequencing will be useful to study strain isolates from a suspicious deliberate contamination event. By applying bioinformatics it is possible to rapidly analyze large amounts of sequence data with minimal post-processing time

To obtain diagnostic bioterrorism response strategies a number of issues must be solved including strategic planning, laboratory infrastructure and standards. The laboratory work has to be strongly linked to different early warning and surveillance systems. Multiple teams as well as joint laboratory protocols are also important resources to have in place. The development of diagnostic bioterrorism response strategies should be based on lessons learned from previous attacks/incidents, planning scenarios, R&D activities and validation and implementations of methods. Laboratories must be able to detect, identify, respond and

recover from covert and overt bioterrorism incidents. Key recommendations include:

Robust sampling and laboratory capacity for high-throughput needs

Laboratory capabilities for diagnostic characterization needs

Multisectoral and international laboratory cooperation to obtain rapid detection and

Writing of this chapter has been supported by grants from the Swedish Civil Contingencies Agency (Anslag 2:4 Krisberedskap), Sweden (Swedish Laboratory Response Network), and the framework of the EU-project AniBioThreat (Grant Agreement:

laboratory (Photo: SVA).

(Segerman et al., 2011).

identification of biothreat agents

Efficient IT-systems for sharing of data

**8. Acknowledgements** 

**7. Conclusions** 

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**5** 

*India* 

**Recent Advancement in the** 

Riyasat Ali and D.N. Rao

**Development of Vaccines Against** *Y. pestis*

*Department of Biochemistry, All India Institute of Medical Sciences, New Delhi,* 

A bioterrorism attack- is the deliberate release of biological agents such as viruses, bacteria, or toxins used to cause illness or death in people, animals, or plants (CDC). These agents are typically found in nature, but it is possible that they could be changed to increase their ability to cause disease, make them resistant to current therapeutics, or to increase their ability to be spread into the environment. Biological agents can be spread through the air, through water, or in food. Terrorists may use biological agents because they can be extremely difficult to detect and may not cause illness for several hours to several days. Some bioterrorism agents, like Variola major and *Yersinia pestis*, can be spread from person to person, while others e.g. *Bacillus anthracis* are not (Bioterrorism review, 2009). Biological agents make attractive weapons because they are relatively easy to obtain and carry from place to place, can be easily dispersed, and can cause widespread fear and panic beyond the actual physical damage they can cause. Many of the agents that could be used for bioterrorism have been divided into three categories A, B, and C, for public health preparedness based on various characteristics of the microbes or the

Category A includes the most "dangerous" and highest priority for public health preparedness. Some of these pathogens can be transmitted from person-to-person, cause diseases with a high mortality rate and are likely to cause public panic and social disruption. Category A agents include *B. anthracis* (anthrax), Variola major (smallpox), *Francisella tularensis* (tularemia), *Y. pestis* (plague), *Clostridium botulinum* neurotoxin (botulism), and Viral hemorrhagic fever viruses (e.g. arenaviruses, filoviruses, bunyaviruses, and

This chapter will discuss the Category A agent *Y.pestis,*the disease it causes,and recent

Plague, a zoonotic disease caused by the gram-negative bacillus *Y. pestis* is primarily a disease of rodents, with transmission occurring through infected fleas. Human disease is acquired through rodent flea vectors, as well as respiratory droplets from animal to humans

**1. Introduction** 

diseases they cause.

flaviviruses).

efforts to develop vaccines.

and humans to humans.

**– A Potential Agent of Bioterrorism** 

