Conceived and designed the experiments: CKM ARH TP PK. Performed the experiments: CKM CAA JB EPP SRW. Analyzed the data: CKM CAA JB EPP SRW TP PK ARH. Contributed reagents/materials/analysis tools: CKM CAA JB EPP SRW TP PK ARH. Wrote the paper: CKM CAA JB EPP SRW TP PK ARH.
The authors have declared that no competing interests exist.
To determine potential links between the clinical isolate to animal products and their geographic origin, we genotyped (MLVA-8, MVLA-15, and canSNP analysis) 80 environmental and 12 clinical isolates and 2 clinical specimens from five cases of anthrax (California in 1976 [n = 1], New York in 2006 [n = 1], Connecticut in 2007 [n = 2], and New Hampshire in 2009[n = 1]) resulting from recreational handling of animal products. For the California case, four clinical isolates were identified as MLVA-8 genotype (GT) 76 and in the canSNP A.Br.Vollum lineage, which is consistent with the Pakistani origin of the yarn. Twenty eight of the California isolates were in the A.Br.Vollum canSNP lineage and one isolate was in the A.Br. 003/004 canSNP sub-group. All 52 isolates and both clinical specimens related to the New York and Connecticut cases were MLVA-8 GT 1. The animal products associated with the NY and CT cases were believed to originate from West Africa, but no isolates from this region are available to be genotyped for comparison. All isolates associated with the New Hampshire case were identical and had a new genotype (GT 149). Isolates from the NY, CT and NH cases diverge from the established canSNP phylogeny near the base of the A.Br.011/009. This report illustrates the power of the current genotyping methods and the dramatically different epidemiological conditions that can lead to infections (i.e., contamination by a single genotype versus widespread contamination of numerous genotypes). These cases illustrate the need to acquire and genotype global isolates so that accurate assignments can be made about isolate origins.
Reports of anthrax cases related to handling of animal products in a non-industrial setting are rare in the USA. In 1974, a cutaneous anthrax case was reported in a woman who bought drums made from goat hides in Haiti
Recently, two inhalation and two cutaneous cases of anthrax were related to drum-making using imported goat hides. In 2006, two unrelated inhalation anthrax cases were reported in New York City and Scotland
While
In this report, we describe the molecular epidemiology of the anthrax cases linked to the recreational use of animal products occurring in the USA which includes the California and New York City inhalation anthrax cases, the two cutaneous cases from Connecticut, and the gastrointestinal case in New Hampshire. In all five cases, epidemiologic and environmental investigations were carried out to determine the source of exposure. For the 2006 and 2007 cases, law enforcement authorities were also involved to rule out any potential criminal activity. Numerous samples were collected from each patient's home, work space, or other associated facilities as part of the respective investigations. Culturing of the samples resulted in the recovery of many
In this report, we analyzed 94 isolates and specimens by MLVA-8, MLVA-15 and canSNP analysis from five inhalation and cutaneous anthrax cases from a weaver (1976), three individuals associated with drum-making (2006, 2007), and a drummer (2009).
The six chromosomal loci of the MLVA-8 scheme were detected in all 29 isolates (
Isolate # | State | Source | pXO1 | pXO2 | CG3 |
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GT |
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4100 | CA | Cerebrum | 141 | 139 | 158 | 162 | 229 | 325 | 538 | 604 | 76 |
4099 | CA | Cerebrum | 141 | 139 | 158 | 162 | 229 | 325 | 538 | 604 | 76 |
4098 | CA | Cerebrum | 141 | 139 | 158 | 162 | 229 | 325 | 538 | 604 | 76 |
4286 | CA | Pleural fluid | 141 | 139 | 158 | 162 | 229 | 325 | 538 | 604 | 76 |
4281 | CA | Pleural fluid | — | — | 158 | 162 | 229 | 325 | 538 | 604 | NA |
4083 | CA | Mediastinal node | — | — | 158 | 162 | 229 | 325 | 538 | 604 | NA |
4085 | CA | Mediastinal node | — | — | 158 | 162 | 229 | 325 | 538 | 604 | NA |
4275 | CA | CSF | — | — | 158 | 162 | 229 | 325 | 538 | 604 | NA |
4274 | CA | CSF | 155 |
— | 158 | 162 | 229 | 313 | 538 | 604 | NA |
9149 | NH | Blood | 126 | 137 | 153 | 153 | 229 | 313 | 568 |
613 | 149 |
0760 | NY | Blood | 123 | 137 | 153 | 162 | 229 | 313 | 604 | 613 | 1 |
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7851 | CT | Biopsy | 123 | 137 | 153 | 162 | 229 | 313 | 604 | 613 | 1 |
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4277 | CA | Yarn | 132 | 139 | 158 | 162 | 229 | 313 | 538 | 604 | 71 |
4276 | CA | Yarn | 132 | 139 | 158 | 162 | 229 | 313 | 538 | 604 | 71 |
4246 | CA | Loom | 129 | 139 | 158 | 162 | 229 | 313 | 538 | 604 | 72 |
4255 | CA | Yarn | 129 | 139 | 158 | 162 | 229 | 313 | 538 | 604 | 72 |
4291 | CA | Camel/Goat hair | 126 | 143 | 158 | 153 | 229 | 313 | 538 | 604 | 105 |
4290 | CA | Yarn | 132 | 139 | 158 | 153 | 229 | 313 | 538 | 604 | 92 |
9147 | NH | Drum | 126 | 137 | 153 | 153 | 229 | 313 | 568 |
613 | 149 |
1028 | NY | Drum | 123 | 137 | 153 | 162 | 229 | 313 | 604 | 613 | 1 |
4111 | CT | Drum | 123 | 137 | 153 | 162 | 229 | 313 | 604 | 613 | 1 |
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4251 | CA | Wool | — | — | 158 | 153 | 229 | 313 | 538 | 604 | NA |
4096 | CA | Yarn | — | — | 158 | 153 | 229 | 313 | 538 | 604 | NA |
4297 | CA | Camel/Goat hair | __ | __ | 158 | 153 | 229 | 301 | 538 | 604 | NA |
4254 | CA | Wool | — | — | 158 | 153 | 229 | 301 | 538 | 604 | NA |
4280 | CA | Wool | — | — | 158 | 153 | 229 | 301 | 538 | 604 | NA |
4273 | CA | Yarn | — | — | 158 | 162 | 229 | 313 | 538 | 604 | NA |
4094 | CA | Yarn | — | — | 158 | 162 | 229 | 313 | 538 | 604 | NA |
4282 | CA | Loom | — | — | 158 | 162 | 229 | 313 | 538 | 604 | NA |
4090 | CA | Yarn | 132 | — | 158 | 153 | 229 | 289 | 538 | 604 | NA |
4093 | CA | Yarn | 132 | — | 158 | 162 | 229 | 313 | 538 | 604 | NA |
4259 | CA | Yarn | 120 | — | 158 | 162 | 229 | 313 | 400 | 532 | NA |
4091 | CA | Yarn | — | 139 | 158 | 162 | 229 | 313 | 538 | 604 | NA |
4095 | CA | Yarn | — | 139 | 158 | 153 | 229 | 301 | 538 | 604 | NA |
4285 | CA | Human hair | — | 139 | 158 | 153 | 229 | 301 | 538 | 604 | NA |
—, plasmid loci not detected.
NA, not applicable.
*, new allele size not previously described by Keim et al.
Among the nine clinical isolates, all four isolates containing both plasmids were identified as GT 76. An additional four isolates were cured of both plasmids but had identical allele sizes for all six chromosomal loci as the GT 76 isolates. However, one isolate, 4274, differed in allele size at the
Of the environmental isolates containing both plasmids (n = 6), five isolates had MLVA-8 genotypes which had been previously described: two yarn isolates were GT 71 and one isolate from the loom and an additional yarn isolate were GT 72. In addition, one isolate (from yarn) was GT 92. One camel/goat hair isolate was GT 105 which has not been previously observed (
All other genotypes are reference genotypes from Keim et al.
With one exception, all of the California isolates were in the A.Br.Vollum lineage by canSNP analysis. One isolate, 4259, was shown to be in the A.Br.003/004 sub-group (
The new branch, “A.Br.011,” is flanked by branches A.Br.008 and A.Br.009. Thus, the group A.Br.008/009 is now subdivided into two groups: A.Br.008/011 and A.Br.011/009. The canSNP signature and assay that defines this new branch is provided in
Sample type | Number | MLVA-8 genotype(s) | Identical by MLVA-15 | CanSNP lineage |
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Clinical isolates |
9 | 76 (n = 4) | NT | A.Br.Vollum (n = 9) |
Environmental isolates |
20 |
71 (n = 2), 72 (n = 2), 92 (n = 1), 105 (n = 1) | NT | A.Br.Vollum (n = 19), A.Br.003/004 (n = 1) |
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Clinical isolate | 1 | 1 | Yes | A.Br.011/009 (n = 1) |
Environmental isolates | 36 | 1 | Yes | A.Br.011/009 (n = 36) |
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Clinical sample |
2 | 1 (n = 1) | Yes | NT |
Environmental isolates | 15 | 1 | Yes | A.Br.011/009 (n = 15) |
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Clinical isolate | 2 | 149 | Yes | A.Br.011/009 (n = 2) |
Environmental isolates | 9 | 149 | Yes | A.Br.011/009 (n = 9) |
NT, not tested.
, Other MLVA-8 genotypes found but could not be assigned due to plasmid loss.
, DNA from biopsy.
,14 isolates lacked one or both virulence plasmids and could not be genotyped.
, canSNP lineage associated with one isolate, 4259.
The clinical isolate from the patient was identified as MLVA-8 GT 1 (
While the biopsy specimen from the drum-maker did not amplify any of the MLVA loci, all MLVA-8 loci were amplified from the biopsy specimen from the drum-maker's son and identified as MLVA-8 GT 1. All environmental isolates from the drum-maker's home and shed where he processed the goat hides were also MLVA-8 GT 1 (
The two clinical isolates from the patient and the nine environmental isolates recovered from the drums and the community building were identified as a new MLVA-8 genotype, designated GT 149 (
Among the five cases reported in this study, we were able to match clinical isolates or samples with environmental isolates in four of the cases (NY case, 2 CT cases, and NH case). Conversely, we were not able to match the clinical and environmental isolates associated with the California case. At the time of the California case (1976), molecular subtyping systems were not available to allow for more precise characterization of the clinical isolates and their possible linkage to environmental contamination. In this report, MLVA was used successfully to detect numerous genotypes in the environment of the California weaver's residence. Although none of the environmental isolate genotypes matched the clinical isolate genotypes, the clinical and environmental isolates that were completely genotyped appear to be closely related within the A4 MLVA cluster (Vollum cluster) as described by Keim et al. (
Based on the number of genotypes found in the environment of the California weaver's home, a co-infection of the patient might be expected. While all the clinical isolates were in the same canSNP lineage, we identified two MLVA-8 genotypes. Four of the isolates were GT 76, and one of the isolates, 4274, differed from the other clinical isolates at two loci demonstrating this isolate differs genotypically from GT 76. In addition, in the original investigation, two different biotypes of
None of the environmental isolates from the California case were an exact match to either clinical isolate genotype. However, several of the environmental isolates (GT 71 and 72) differed from the clinical isolates only at the pXO1 loci. Thus, the matching genotype among the environmental isolates may not have been identified due to the high rate of pXO1 loss in our archived isolates associated with this case. The isolates included in this report from the California case were stored for over 25 years at room temperature on agar slants with mineral oil overlay. We have previously documented that the storage conditions used for these isolates may have had an adverse effect on their plasmid stability and, thus, caused the loss of plasmids over the course of their long-term storage
In contrast to the California case where multiple MLVA genotypes were found, the isolates collected in the NY, CT and NH cases were MLVA-8 GT 1 (NY and CT) and MLVA-8 GT 149 (NH), and all isolates from these three cases belong in the A.Br.011/009 lineage. The animal products associated with the NY and CT cases were believed to originate from West Africa, but only isolates from other African regions were available to be genotyped for comparison. The link between the canSNP lineage and the West African origin of the hides is surprising as only one isolate originating from Africa (isolated in Ethiopia) has been discovered to belong in this canSNP A.Br.011/009 lineage but possesses a different MLVA genotype. While molecular subtyping data exists for isolates from the southern parts of Africa, very little information is known regarding the molecular subtypes of isolates in the western region of Africa. Molecular subtyping of additional
While little is known regarding the diversity of
Similar to the NY and CT cases, all of the isolates associated with the NH case were identical and MLVA-8 was used to successfully match the clinical and environmental isolates from this case. However, the NH isolates were a new genotype, GT 149. While this genotype differed from GT 1 at three loci (vrrB2, vrrC1, pXO1), it was within the same cluster (A1.a) as GT 1 (
In the current report, we used MLVA and canSNP analysis of
This report does not meet the definition of research under 45 CFR 46.102(d), the U. S. Code of Federal Regulations regarding the protection of human subjects. Thus, review from our institutional review board was not required, and a waiver was obtained. Specimens were initially obtained during the course of outbreak investigations and submitted for diagnostic testing; specific informed consent was not obtained, although individuals were free to decline. Activities that do not meet the definition of research are not subject to informed consent requirements under 45 CFR 46.
Twenty nine isolates related to the California inhalation case were included in this report (
The clinical isolate from the patient and 36 environmental isolates were included from the 2006 New York City case. The environmental isolates were recovered from samples collected in the drum-maker's warehouse and home, from goat hides in the drum-maker's possession, and in the vehicle that was used to transport the goat hides.
No clinical isolates were recovered from the 2007 cutaneous anthrax cases from Connecticut. However, biopsy specimens from both patients were tested. In addition, 15 environmental isolates recovered from samples taken in the drum-maker's residence and work shed, previously made drums, vehicle and goat hides found in the work shed were included in this report.
Eleven isolates associated with the 2009 New Hampshire case were included in the report. Two clinical isolates from the patient and nine environmental isolates that were recovered from drums and the community building where the patient attended the drumming event were assayed.
Isolates were stored in 25% glycerol with water and held at −70°C until needed. The clinical specimens were stored at −20°C until processed. Prior to their inclusion in this study, the California isolates were stored at ambient temperature on tryptic soy agar slants overlayed with mineral oil in various physical locations for over 25 years until they were recovered from the slants in 2002
DNA template from all recovered isolates was obtained by heat-lysis of a single colony after overnight growth on SBA. Using a 1 µl loop, one colony was suspended in 200 µl of 10 mM Tris-HCl pH 8.0 in a 1.5-ml tube containing a 0.22 µM filter unit (Millipore, Billerica, MA). The suspension was heated at 95°C for 20 min and centrifuged in a microfuge at 6000× g for 2 min. The filter unit was discarded, and the cell lysate was held at −20°C until testing. One µl of lysate was used in each reaction.
DNA from clinical specimens was extracted using the QIAamp DNA Mini kit (Qiagen, Valencia, CA) according to manufacturer's instructions.
MLVA-8 and the expanded MLVA-15 were performed as previously described by Keim et al. and Van Ert et al., respectively
CanSNP analysis was performed as previously described
Assay name | A.Br.011 | A.Br.011/009_3692595 |
Assay target | Provides resolution within A.Br.008/009 group | SNP on A.Br.011/009 branch (terminal genome is A0343) |
SNP Position (bp) |
2552486 | 3692595 |
Ancestral primer (5′-3′) |
AAACGAATTCCCGCTGAAAATAcT |
CCCTAAAAAAGCAGAGACTATg |
Derived primer (5′-3′) |
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Consensus primer (5′-3′) |
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Assay format |
meltMAMA | SYBR MAMA |
Using ‘Ames ancestor’ genome (GenBank ref: AE017334).
Underlined nucleotides indicate the position of the SNP; bolded nucleotides indicate an introduced GC clamp that increases the melt temperature of the primer, thus enhancing allelic discrimination
All assays were optimized on an Applied Biosystems ABI PRISM 7900HT Sequence Detection System using default thermocycling parameters, with the addition of the dissociation curve
We would like to acknowledge Richard Okinaka, Dawn Birdsell, Molly Matthews and James Schupp, who assisted with assay design and genotyping of some of the samples used in this report.