Genotype Analysis of Bacillus anthracis Strains Circulating in Bangladesh

In Bangladesh, anthrax, caused by the bacterium Bacillus anthracis, is considered an endemic disease affecting ruminants with sporadic zoonotic occurrences in humans. Due to the lack of knowledge about risks from an incorrect removal of infected carcasses, the disease is not properly monitored, and because of the socio-economic conditions, the situation is under-reported and under-diagnosed. For sensitive species, anthrax represents a fatal outcome with sudden death and sometimes bleeding from natural orifices. The most common source of infection for ruminants is ingestion of spores during grazing in contaminated pastures or through grass and water contaminated with anthrax spores. Domestic cattle, sheep and goats can also become infected through contaminated bone meal (used as feed) originating from anthrax-infected carcasses. The present investigation was conducted to isolate B. anthracis organisms from 169 samples (73 soil, 1 tissue, 4 bone and 91 bone meal samples) collected from 12 different districts of Bangladesh. The sampling was carried out from 2012 to 2015. Twelve samples resulted positive for B. anthracis. Biomolecular analyses were conducted starting from the Canonical Single Nucleotide Polymorphism (CanSNP) to analyze the phylogenetic origin of strains. The analysis of genotype, obtained through the Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) with the analysis of 15 Variable Number Tandem Repeats (VNTR), demonstrated four different genotypes: two of them were previously identified in the district of Sirajganj. The sub-genotyping, conducted with Single Nucleotide Repeats analysis, revealed the presence of eight subgenotypes. The data of the present study concluded that there was no observed correlation between imported cattle feed and anthrax occurrence in Bangladesh and that the remarkable genetic variations of B. anthracis were found in the soil of numerous outbreaks in this country.     

Use of Repetitive DNA Sequences and the PCR To Differentiate Escherichia coli Isolates from Human and Animal Sources

The rep-PCR DNA fingerprint technique, which uses repetitive intergenic DNA sequences, was investigated as a way to differentiate between human and animal sources of fecal pollution. BOX and REP primers were used to generate DNA fingerprints from Escherichia coli strains isolated from human and animal sources (geese, ducks, cows, pigs, chickens, and sheep). Our initial studies revealed that the DNA fingerprints obtained with the BOX primer were more effective for grouping E. coli strains than the DNA fingerprints obtained with REP primers. The BOX primer DNA fingerprints of 154 E. coli isolates were analyzed by using the Jaccard band-matching algorithm. Jackknife analysis of the resulting similarity coefficients revealed that 100% of the chicken and cow isolates and between 78 and 90% of the human, goose, duck, pig, and sheep isolates were assigned to the correct source groups. A dendrogram constructed by using Jaccard similarity coefficients almost completely separated the human isolates from the nonhuman isolates. Multivariate analysis of variance, a form of discriminant analysis, successfully differentiated the isolates and placed them in the appropriate source groups. Taken together, our results indicate that rep-PCR performed with the BOX A1R primer may be a useful and effective tool for rapidly determining sources of fecal pollution.     

Strategy for Identification of Bacillus cereus and Bacillus thuringiensis Strains Closely Related to Bacillus anthracis

Bacillus cereus strains that are genetically closely related to B. anthracis can display anthrax-like virulence traits (A. R. Hoffmaster et al., Proc. Natl. Acad. Sci. USA 101:8449-8454, 2004). Hence, approaches that rapidly identify these “near neighbors” are of great interest for the study of B. anthracis virulence mechanisms, as well as to prevent the use of such strains for B. anthracis-based bioweapon development. Here, a strategy is proposed for the identification of near neighbors of B. anthracis based on single nucleotide polymorphisms (SNP) in the 16S-23S rRNA intergenic spacer (ITS) containing tRNA genes, characteristic of B. anthracis. By using restriction site insertion-PCR (RSI-PCR) the presence of two SNP typical of B. anthracis was screened in 126 B. cereus group strains of different origin. Two B. cereus strains and one B. thuringiensis strain showed RSI-PCR profiles identical to that of B. anthracis. The sequencing of the entire ITS containing tRNA genes revealed two of the strains to be identical to B. anthracis. The strict relationship with B. anthracis was confirmed by multilocus sequence typing (MLST) of four other independent loci: cerA, plcR, AC-390, and SG-749. The relationship to B. anthracis of the three strains described by MLST was comparable and even higher to that of four B. cereus strains associated with periodontitis in humans and previously reported as the closest known strains to B. anthracis. SNP in ITS containing tRNA genes combined with RSI-PCR provide a very efficient tool for the identification of strains closely related to B. anthracis.     

Identification of Bacillus anthracis Spore Component Antigens Conserved across Diverse Bacillus cereus sensu lato Strains

We sought to identify proteins in the Bacillus anthracis spore, conserved in other strains of the closely related Bacillus cereus group, that elicit an immune response in mammals. Two high throughput approaches were used. First, an in silico screening identified 200 conserved putative B. anthracis spore components. A total of 192 of those candidate genes were expressed and purified in vitro, 75 of which reacted with the rabbit immune sera generated against B. anthracis spores. The second approach was to screen for cross-reacting antigens in the spore proteome of 10 diverse B. cereus group strains. Two-dimensional electrophoresis resolved more than 200 protein spots in each spore preparation. About 72% of the protein spots were found in all the strains. 18 of these conserved proteins reacted against anti-B. anthracis spore rabbit immune sera, two of which (alanine racemase, Dal-1 and the methionine transporter, MetN) overlapped the set of proteins identified using the in silico screen. A conserved repeat domain protein (Crd) was the most immunoreactive protein found broadly across B. cereus sensu lato strains. We have established an approach for finding conserved targets across a species using population genomics and proteomics. The results of these screens suggest the possibility of a multiepitope antigen for broad host range diagnostics or therapeutics against Bacillus spore infection.The anthrax causing bacterium Bacillus anthracis is a member of the Bacillus cereus sensu lato (s.l.)¹ group, a term given to the polyphyletic species consisting of Bacillus thuringiensis, Bacillus cereus, Bacillus mycoides, Bacillus weihenstephanensis, and Bacillus pseudomycoides (1). Genomics studies of B. cereus s.l. strains have shown a similar chromosomal gene composition within this group (2–7). Many phenotypes that distinguish B. cereus s.l. members, such as crystalline toxin production (8), emesis in humans (9), and anthrax virulence (10), are encoded by genes on large plasmids. Experimental conjugative transfer of plasmids between B. cereus s.l. strains has been demonstrated in vitro, in complex media, and in vector species (11–13). Therefore there is a concern about transfer of virulence genes between genetic backgrounds creating new pathogen lineages. In this regard, there is an emerging evidence of natural dissemination of the pXO1 and pXO2 plasmids that encode the anthrax lethal toxin and capsule, respectively. For example, B. cereus G9241 carries a pXO1 plasmid and lethal toxin genes almost identical to those in B. anthracis (6), and a B. cereus strain, which causes anthrax-like illness in African great apes, apparently contains both pXO1 and pXO2 plasmids (14).The infectious agent of most if not all human B. cereus s.l. diseases is the spore. The spore is a dormant, environmentally resistant structure that persists in nutrient- or water-limiting conditions. Anthrax infection occurs after introduction of the B. anthracis spore into a skin abrasion or via inhalation or ingestion (10). The spore germinates inside host cells, and the resulting vegetative bacteria express toxins and capsules that elicit an immune response (10, 15, 16). Formation of the B. cereus spore involves asymmetric cell division during which a copy of the genome is partitioned into each of the sister cells. The smaller cell (prespore) develops into mature endospore, and the larger cell (mother cell) contributes to the differentiation process but undergoes autolysis following its completion to release the endospore into the surrounding medium. Synthesis of cortex, coat, and exosporium are a function mainly of the mother cell. The cortex and coat layers are in close proximity to one another, whereas the exosporium tends to appear as an irregularly shaped, loosely attached, balloon-like layer (17–20). The coat and the exosporium contribute to the remarkable resistance of spores to extreme physical and chemical stresses including the exposure to extraterrestrial conditions (21, 22). Recent work on the structure, composition, assembly, and function of the spore coat and exosporium of pathogenic organisms like B. anthracis and B. cereus have highlighted the crucial link that exists between the origin of these layers (19, 23). There are differences in the appearance and thickness of the coat layers among the spores of various strains and species. In some B. thuringiensis strains, the inner coat is laminated but consists of a patchwork of striated packets, appearing either stacked or comblike, and the outer coat is granular (24), whereas in B. anthracis and other B. cereus s.l. isolates the coat appears compact (25–27). The coat layers comprise about 30% of the total proteins present in the spore (19, 28). Intraspecies variation in the structure and composition of the spore surface layers may reflect the environmental conditions under which these spores are formed (29–31).Because the spore is crucial to infection and persistence of B. anthracis and its close relatives, we undertook an investigation of its protein profile variability across the B. cereus s.l. group. Our goal in this study was to identify conserved antigenic spore proteins that may be transitioned in the future as candidates for immunodiagnostics, therapeutics, or vaccines. We used two high throughput approaches: genome-based bioinformatics analysis and comparative proteomics analysis of spores of B. cereus s.l. to select conserved targets. Our analysis revealed a list of conserved spore proteins within B. cereus but relatively few cross-reacting antigens. Two of these spore conserved antigens (Crd and MetN) have not been described previously for B. anthracis.     

Diversity of Bacillus anthracis Strains in Georgia and of Vaccine Strains from the Former Soviet Union

Despite the increased number of anthrax outbreaks in Georgia and the other Caucasian republics of the former Soviet Union, no data are available on the diversity of the Bacillus anthracis strains involved. There is also little data available on strains from the former Soviet Union, including the strains previously used for vaccine preparation. In this study we used eight-locus variable-number tandem repeat analyses to genotype 18 strains isolated from infected animals and humans at different sites across Georgia, where anthrax outbreaks have occurred in the last 10 years, and 5 strains widely used for preparation of human and veterinary vaccines in the former Soviet Union. Three different genotypes affiliated with the A3.a cluster were detected for the Georgian isolates. Two genotypes were previously shown to include Turkish isolates, indicating that there is a regional strain pattern in the South Caucasian-Turkish region. Four of the vaccine strains were polymorphic, exhibiting three different patterns of the cluster A1.a genotype and the cluster A3.b genotype. The genotype of vaccine strain 71/12, which is considered an attenuated strain in spite of the presence of both of the virulence pXO plasmids, appeared to be a novel genotype in the A1.a cluster.     

Use of the Chrome Azurol S Agar Plate Technique To Differentiate Strains and Field Isolates of Rhizobium leguminosarum biovar trifolii

Identification of Rhizobium and Bradyrhizobium strains and especially of indigenous isolates continues to be one of the major difficulties associated with competition studies. Because there is no universally accepted method, the method of choice depends on preference, experience, and equipment. Here, an agar plate technique was used to distinguish strains and field isolates of Rhizobium leguminosarum biovar trifolii to provide a basis for identifying nodule occupants in further competition studies. A rapid plate technique, based on differential growth characteristics, complements other techniques such as serological reactions, particularly when antisera cross-react with nonhomologous strains. The technique involves culturing strains and isolates on chrome azurol S agar. Although similar responses were observed among some strains, the response was highly reproducible and was considered an ideal complementary technique used in conjunction with serological procedures. Strains with similar responses could often be differentiated by varying media components, such as the source of carbon.     

Comparison of Growth and Toxin Production in Two Vaccine Strains of Bacillus anthracis

Two vaccine strains of Bacillus anthracis were monitored in a 10-liter fermentor to compare growth patterns and toxin production. Under identical conditions, the Sterne strain produced all three components of anthrax toxin, whereas strain V770 produced only the protective antigen.     

Identification by Quantitative Carrier Test of Surrogate Spore-Forming Bacteria To Assess Sporicidal Chemicals for Use against Bacillus anthracis

The spores of six strains of Bacillus anthracis (four virulent and two avirulent) were compared with those of four other types of spore-forming bacteria for their resistance to four liquid chemical sporicides (sodium hypochlorite at 5,000 ppm available chlorine, 70,000 ppm accelerated H₂O₂, 1,000 ppm chlorine dioxide, and 3,000 ppm peracetic acid). All test bacteria were grown in a 1:10 dilution of Columbia broth (with manganese) incubated at 37°C for 72 h. The spore suspensions, heat treated at 80°C for 10 min to rid them of any viable vegetative cells, contained 1 × 10⁸ to 3 × 10⁸ CFU/ml. The second tier of the quantitative carrier test (QCT-2), a standard of ASTM International, was used to assess for sporicidal activity, with disks (1 cm in diameter) of brushed and magnetized stainless steel as spore carriers. Each carrier, with 10 μl (≥10⁶ CFU) of the test spore suspension in a soil load, was dried and then overlaid with 50 μl of the sporicide being evaluated. The contact time at room temperature ranged from 5 to 20 min, and the arbitrarily set criterion for acceptable sporicidal activity was a reduction of ≥10⁶ in viable spore count. Each test was repeated at least three times. In the final analysis, the spores of Bacillus licheniformis (ATCC 14580^T) and Bacillus subtilis (ATCC 6051^T) proved to be generally more resistant than the spores of the strains of B. anthracis tested. The use of one or both of the safe and easy-to-handle surrogates identified here should help in developing safer and more-effective sporicides and also in evaluating the field effectiveness of existing and newer formulations in the decontamination of objects and surfaces suspected of B. anthracis contamination.     

Use of a Plasmid DNA Probe To Monitor Populations of Bacillus pumilus Inoculant Strains in Hay

We are evaluating naturally occurring isolates of Bacillus pumilus for use as microbial hay preservatives. Seven isolates of B. pumilus from hay contained a 42-kb cryptic plasmid (pMGD296). We wished to determine whether pMGD296 could be used as a molecular marker to follow populations of these isolates in hay over time. Southern blots and colony blots of 69 isolates of B. pumilus and other Bacillus spp. were probed with ³²P-labeled pMGD296. Twenty-nine probe-positive isolates were identified; of these, 28 contained a plasmid with a restriction profile identical to that of pMGD296. One isolate from untreated hay contained a 40-kb plasmid (pMGD150) that was homologous to pMGD296 but had a different restriction fragment pattern. Regions of homology between the two plasmids were identified by Southern blotting, and a 1.9-kb HindIII-PstI fragment of pMGD296 lacking strong homology to pMGD150 was cloned in pUC18. The cloned fragment hybridized only with isolates containing pMGD296 and was used to estimate populations of these isolates in treated and untreated hay.     

A Novel Multiplex PCR Discriminates Bacillus anthracis and Its Genetically Related Strains from Other Bacillus cereus Group Species

Anthrax is an important zoonotic disease worldwide that is caused by Bacillus anthracis, a spore-forming pathogenic bacterium. A rapid and sensitive method to detect B. anthracis is important for anthrax risk management and control in animal cases to address public health issues. However, it has recently become difficult to identify B. anthracis by using previously reported molecular-based methods because of the emergence of B. cereus, which causes severe extra-intestinal infection, as well as the human pathogenic B. thuringiensis, both of which are genetically related to B. anthracis. The close genetic relation of chromosomal backgrounds has led to complexity of molecular-based diagnosis. In this study, we established a B. anthracis multiplex PCR that can screen for the presence of B. anthracis virulent plasmids and differentiate B. anthracis and its genetically related strains from other B. cereus group species. Six sets of primers targeting a chromosome of B. anthracis and B. anthracis-like strains, two virulent plasmids, pXO1 and pXO2, a bacterial gene, 16S rRNA gene, and a mammalian gene, actin-beta gene, were designed. The multiplex PCR detected approximately 3.0 CFU of B. anthracis DNA per PCR reaction and was sensitive to B. anthracis. The internal control primers also detected all bacterial and mammalian DNAs examined, indicating the practical applicability of this assay as it enables monitoring of appropriate amplification. The assay was also applied for detection of clinical strains genetically related to B. anthracis, which were B. cereus strains isolated from outbreaks of hospital infections in Japan, and field strains isolated in Zambia, and the assay differentiated B. anthracis and its genetically related strains from other B. cereus group strains. Taken together, the results indicate that the newly developed multiplex PCR is a sensitive and practical method for detecting B. anthracis.     

Use of superabsorbent polymer gels for surface decontamination of Bacillus anthracis spores

Aims:  This study evaluated the inactivation of Bacillus anthracis Vollum spores dried on a nonporous surface using a superabsorbent polymer (SAP) gel containing commercially available liquid decontaminants.
Methods and Results:  The first phase determining the availability of the liquid decontaminant within the SAP showed that the SAP gel containing pH-adjusted sodium hypochlorite (NaOCl) inhibited B. anthracis growth while the water control SAP gel had no affect on growth. For testing surface decontamination, B. anthracis spores were dried onto steel coupons painted with chemical agent resistant coating and exposed to SAP containing either pH-adjusted NaOCl, chlorine dioxide (ClO₂) or hydrogen peroxide/peracetic acid (H₂O₂/PA) for 5 and 30 min. At contact times of both 5 and 30 min, all of the SAP gels containing pH-adjusted NaOCl, ClO₂ or H₂O₂/PA inactivated B. anthracis spores at levels ranging from 2·2 to ≥7·6 log reductions.
Conclusions:  Incorporation of three commercially available decontaminant technologies into a SAP gel promotes inactivation of B. anthracis spores without observable physical damage to the test surface.
Significance and Impact of the Study:  This work provides preliminary data for the feasibility of using SAP in inactivating B. anthracis spores on a nonporous surface, supporting the potential use of SAP in surface decontamination.     

Distribution of the Insertion Element IS240 Among Bacillus thuringiensis Strains

The presence of IS240 was investigated in 69 Bacillus thuringiensis (Bt) strains including strains from serotype H1 to H45 and additional strains with known Dipteran larvae toxicity. Restriction digests of total DNA and PCR products obtained with a single 16-bases primer corresponding to the IS240 inverted repeated sequence were hybridized with the IS240A element. The results indicate that 67% of the Bt strains tested, including all known mosquitocidal strains, possess at least one IS240-related element. PCR experiments indicate that IS240 represents a family of insertion sequences with several variants. Received: 15 October 1996 / Accepted: 20 November 1996     

Use of long-range repetitive element polymorphism-PCR to differentiate Bacillus anthracis strains.

The genome of Bacillus anthracis is extremely monomorphic, and thus individual strains have often proven to be recalcitrant to differentiation at the molecular level. Long-range repetitive element polymorphism-PCR (LR REP-PCR) was used to differentiate various B. anthracis strains. A single PCR primer derived from a repetitive DNA element was able to amplify variable segments of a bacterial genome as large as 10 kb. We were able to characterize five genetically distinct groups by examining 105 B. anthracis strains of diverse geographical origins. All B. anthracis strains produced fingerprints comprising seven to eight bands, referred to as "skeleton" bands, while one to three "diagnostic" bands differentiated between B. anthracis strains. LR REP-PCR fingerprints of B. anthracis strains showed very little in common with those of other closely related species such as B. cereus, B. thuringiensis, and B. mycoides, suggesting relative heterogeneity among the non-B. anthracis strains. Fingerprints from transitional non-B. anthracis strains, which possessed the B. anthracis chromosomal marker Ba813, scarcely resembled those observed for any of the five distinct B. anthracis groups that we have identified. The LR REP-PCR method described in this report provides a simple means of differentiating B. anthracis strains.     

Behavior of Bacillus anthracis Strains Sterne and Ames K0610 in Sterile Raw Ground Beef

The behavior of Bacillus anthracis Sterne spores in sterile raw ground beef was measured at storage temperatures of 2 to 70°C, encompassing both bacterial growth and death. B. anthracis Sterne was weakly inactivated (−0.003 to −0.014 log₁₀ CFU/h) at storage temperatures of 2 to 16°C and at temperatures greater than and equal to 45°C. Growth was observed from 17 to 44°C. At these intermediate temperatures, B. anthracis Sterne displayed growth patterns with lag, growth, and stationary phases. The lag phase duration decreased with increasing temperature and ranged from approximately 3 to 53 h. The growth rate increased with increasing temperature from 0.011 to 0.496 log₁₀ CFU/h. Maximum population densities (MPDs) ranged from 5.9 to 7.9 log₁₀ CFU/g. In addition, the fate of B. anthracis Ames K0610 was measured at 10, 15, 25, 30, 35, 40, and 70°C to compare its behavior with that of Sterne. There were no significant differences between the Ames and Sterne strains for both growth rate and lag time. However, the Ames strain displayed an MPD that was 1.0 to 1.6 times higher than that of the Sterne strain at 30, 35, and 40°C. Ames K0610 spores were rapidly inactivated at temperatures greater than or equal to 45°C. The inability of B. anthracis to grow between 2 and 16°C, a relatively low growth rate, and inactivation at elevated temperatures would likely reduce the risk for recommended ground-beef handling and preparation procedures.     

Fingerprinting of Bacillus thuringiensis Type Strains and Isolates by Using Bacillus cereus Group-Specific Repetitive Extragenic Palindromic Sequence-Based PCR Analysis

A total of 119 Bacillus thuringiensis strains (83 type strains and 26 native isolates), as well as five B. cereus group species, were analyzed by repetitive extragenic palindromic sequence-based PCR analysis (Rep-PCR) fingerprinting. Primers Bc-REP-1 and Bc-REP-2 were specifically designed according to an extragenic 26-bp repeated sequence found in the six B. cereus group genomes reported. A total of 47 polymorphic bands were detected, and the patterns varied from 5 to 13 bands in number and from 0.2 to 3.8 kb in size. Virtually each type strain showed a distinctive B. cereus (Bc)-Rep-PCR pattern, except for B. thuringiensis serovars dakota (H serotype 15 [H15]) and sotto (H4a,4b), as well as serovars amagiensis (H29) and seoulensis (H35), which shared the same patterns. As expected, serovar entomocidus (H6) and its biovar subtoxicus showed an identical pattern; similarly, serovars sumiyoshiensis (H3a,3d) and fukuokaensis (H3a,3d,3e), which share two antigenic determinants, also showed identical Bc-Rep-PCR patterns. Interestingly, serovars israelensis (H14) and malaysiensis (H36), which share several phenotypic attributes, also showed identical Bc-Rep-PCR patterns. Native, coleopteran-active strains, including the self-agglutinated LBIT-74 strain, showed Bc-Rep-PCR patterns identical or very similar to that of the tenebrionis strain. Likewise, native mosquitocidal strains (including some self-agglutinated strains) also showed patterns identical or very similar to that of the serovar israelensis IPS-82 strain. Additionally, native β-exotoxin-producing strains from serovar thuringiensis showed patterns identical to that of the B. thuringiensis type strain. The B. cereus group-specific Bc-Rep-PCR fingerprinting technique was shown to be highly discriminative, fast, easy, and able to identify B. thuringiensis serotypes, including nonflagellar and self-agglutinated strains.     

Germination of Bacillus anthracis spores

The influence of amino acids, nucleosides and inorganic components on the kinetics and effectiveness of the germination of B. anthracis spores was studied. The study revealed that the rapid germination of the spores took place after their activation at 65 degrees C in tris buffer with L-alanine in combination with inosine or adenosine added; less pronounced germinative action was caused by the addition of alanine only and the combination of phenylalanine, tyrosine and tryptophan. The rapidity of germination and the sets of effective germinants for spores of different strains were different. All B. anthracis strains under study had nucleotide sequences, of gene gerX in their genome.     

The ecology of Bacillus anthracis

The global distribution of anthrax is largely determined by soils with high calcium levels and a pH above 6.1, which foster spore survival. It is speculated that the spore exosporium probably plays a key part by restricting dispersal and thereby increasing the probability of a grazing animal acquiring a lethal dose. ‘Anthrax Seasons’ are characterized by hot-dry weather which stresses animals and reduces their innate resistance to infection allowing low doses of spores to be infective. Necrophagic flies act as case-multipliers and haemophagic flies as space-multipliers; the latter are aided by climatic factors which play a key part in whether epidemics occur. Host death is a function of species sensitivity to the toxins. The major function of scavengers is to open the carcass, spill fluids, and thereby aid bacilli dispersal and initiate sporulation. In the context of landscape ecology viable spore distribution is a function of mean annual temperature, annual precipitation, elevation, mean NDVI, annual NDVI amplitude, soil moisture content, and soil pH.     

The surface of Bacillus anthracis

Bacillus anthracis is a Gram positive organism possessing a complex parietal structure. An S-layer, a bi-dimensional crystalline layer, and a peptidic capsule surround the thick peptidoglycan of bacilli harvested during infection. A review of the current literature indicates that elements from each of these three structures, as well as membrane components, have been studied. So-called cell-wall secondary polymers, be they attached to the cell-wall or to the membrane play important functions, either per se or because they permit the anchoring of proteins. Some surface proteins, whichever compartment they are attached to, play, as had been hypothesized, key roles in virulence. Others, of yet unknown function, are nevertheless expressed in vivo. This review will focus on well-studied polymers or proteins and indicate, when appropriate, the mechanisms by which they are targeted to their respective locations.