# Presentation: 812892

## ANTHRAXEpidemiology 101

## Bacillus anthracis

- Disease known for thousands of years.
- Organism first described in mid-1800s
- Used as proof of Henle’s postulates by Koch.
- First vaccines developed by Greenfield and Toussaint; public demonstration by Pasteur in 1881.
- Livestock and human vaccines.

**Notes:**
Present research would indicate that this pathogen probably first emerged in Africa in the region of the Khoi-San; that is from the Cape of Good Hope to the Horn of Africa as a disease of wildlife. With domestication of ruminants and the entry of cattle into Africa it was transferred to Eygpt in time for the Vth Plague (1414BC) and by coastal shipping and trade into the Middle East, Central Asia, and Europe.

Davaine & Rayer described anthrax bodies in infected sheep blood in 1850. But Delafond saw “little rods” in 1838. There are other claims for 1849 (Pollander) and 1842 (Fuchs).

Greenfield in England and Toussaint in France developed experimental vaccines. Without government support Greenfield’s vaccine fell out of sight. Toussaint successfully vaccinated sheep with defrinated blood with B. anthracis that had been heat-treated for 10 min. at 55’C.  Pasteur having made a claim for his successful vaccine in fact covertly used Toussaint’s vaccine in his public demonstration of 1881 in Pouilly-le-Fort. It was only later replaced by his vaccine which was then used worldwide for many years. This vaccine resulted from culturing B. anthracis at 42’ to 43’C for 15-20 days; the second innoculum was from a 10-12 day culture. It was not without risk of killing livestock being a mixture of fully pathogenic organisms and capsule –ve organisms.

Present vaccines are the result of Max Sterne’s work in Natal in the late 1930s which lacks the capsule plasmid. The independently developed Russian STI is a Sterne-type vaccine. Human vaccines are essentially ‘Protective Antigen in an AlOH adjuvent.

## Aerobic, sporeforming, Gram-positive capsulated rod
It survives by killing.
The spore has a capacity to survive in the environment for decades.
It is a junior relative of B. cereus/thuringensis.
Pathogenicity depends on two plasmids, pX01 & pX02.

***Bacillus anthracis***

- Aerobic, sporeforming, Gram-positive capsulated rod
- It survives by killing.
- The spore has a capacity to survive in the environment for decades.
- It is a junior relative of *B. cereus/thuringensis.*
- Pathogenicity depends on two plasmids, pX01 & pX02.

## B. anthracis

- PCR analysis utilizes **V**ariable **N**umber **T**andem **R**epeats ... these are repetitive sequence motifs.
- Eight VNTRs are presently used: vrrA, vrrB1, vrrB2, vrrC1, vrrC2, CG3 and one for each plasmid; 35 more are under development.
- Used rather like bar-codes.

## B. anthracis

- Using multiple locus variable tandem repeat analysis we have identified some 89 strains with more awaiting definition.
- The ‘B’ strains are essentially limited to southern Africa.
- The ‘A’ strains are found worldwide.

## Toxin production

- Protective antigen (PA) binds to host cell receptor
- Furin cleaves and releases PA20
- PA63 forms a heptamer
- The toxin enzymes bind to PA63
- Receptor-mediated endocytosis
- Endosome acidification leads to membrane insertion of PA63
- Translocation of toxic enzymes into the cytosol; LF, lethal factor; OF, oedema factor
*Nature* (1997) 385:833-838

## Slide 7

## Slide 8

## ‘HYPOACUTE’

**‘****HYPOACUTE’**

## Cutaneous anthrax

- This is a pathognomic  skin lesion ... with a raised vesiculated edge, inflamed, and with a black base to the ulcer, e.g., *charbon*, ‘Siberian ulcer’.

## Cutaneous anthrax

- This postman had borrowed his son’s woolen scarf; the son worked in a bone meal plant.
- Without prompt treatment, it carries a 10% case fatality rate.

## Slide 12

## Cutaneous anthrax

- Cutaneous lesions follow occupational exposure.
- Usually seen in men after butchering an affected cow; or from handling hides, especially sun-dried hides; or from carrying contaminated building insulation.
- Also following insect bites.

## HYPOACUTE

**HYPOACUTE**

## Slide 15

## 

Onset usually 2-5 days after eating contaminated food
Nausea, fever, malaise, abdominal pain, bloody vomiting & diarrhea
Often fatal even with treatment

**HYPOACUTE**

**GASTROENTERIC**

-  Onset usually 2-5 days after eating contaminated food
-  Nausea, fever, malaise, abdominal pain, bloody vomiting & diarrhea
-  Often fatal even with treatment

- Onset usually 2-5 days after eating contaminated food
- Nausea, fever, malaise, abdominal pain, bloody vomiting & diarrhea
- Often fatal even with treatment

- ( 5 )

## Gastroenteric anthrax

- Seen only in poor, developing countries with food shortages or inadequate veterinary inspection – recent cases in SubSahelian Africa, Central Asia, Russia, India & Thailand.
- Usually have concurrent cutaneous cases from butchering the affected animal or handling the infected meat.
- Probable frequency: one outbreak per 64 infected animals eaten.

## Anthrax status, 1998

**Anthrax status, 1998**

- Not reported

- Sporadic
- Endemic
- Hyperendemic

- Possibly free
- Free (= _>_ 8 years
- without cases)

## Sverdlovsk incident (1979)

- 120 to >400 became ill, 75 died (at least, probably ~ 90)
- Soviet officials initially reported that outbreak was due to contaminated meat
- Autopsy findings of hemorrhagic mediastinitis
- Infection later linked to an aerosol leak from a nearby biologic weapons facility operated by the Soviet army
- Lines of equal dosage based on northerly wind pattern

- Meselson et al, *Science*, 1994

## Sverdlovsk: Anthrax case onsets

## Sverdlovsk: Anthrax deaths

## Sverdlovsk: Case onsets & Deaths

## Sverdlovsk: Residence distances (m) from source

## Sverdlovsk: Work place distances (m) from source

## Sverdlovsk: All distances (m) from source

## Antibiotic resistance & sensitivities

| Sample Id | P | VA5 | VA30 | CIP | SXT | CZ30 | E15 | TE30 | CC2 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| A0850 | S | 4 | R | R | 12 | 8 | 8 | 8 | 9 |
| A0856 | S | R | 6 | 10 | R | 10 | 6 | 8 | 6 |
| A0867 | S | R | 5 | 11 | R | 12 | R | R | 5 |
| Kudu ‘93 | S | 3 | R | 6 | R | 12 | 6 | 9 | 6 |

- Kudu ’93 is our routine reference strain. The others were the three -phage resistant samples in our Italian collection (n=53).
**P** Penicillin; **VA** Vancomycin 5 & g; **CIP** Ciprofloxcin 5g; **SXT** Sulfathoxaxole 23.75g & Trimethoprim 1.25g; **CZ30** Cefazolin 30g; **E15** Erythromycin 15g ; **TE30** Tetracycline 30g ; **CC2** Clindamycin g

**Notes:**
Antibiotic resistance & sensitivities

Normally we only check for penicillin and gamma-phage sensitivities. However when processing these Italian isolates from their IZS archive in Foggia, three isolates were noted to be phage resistant. We infrequently note any phage resistance.

On the spur of the moment my graduate student, decided to check their antibiotic resistance across this wide range, with the preliminary results noted here.

The numbers refer to the distance in mm from the outer edge of the disk to viable colonies; or the radius of the clear zone minus the radius of the antibiotic disk.

A0850 	#1180	GT 1	Sicily	sheep	1996/97			ex IZS Puglia & Basilicata
A0856	#Binosi	GT 1	Puglia	sheep	before 10 Nov 1984	ex IZS Puglia & Basilicata; freeze dried & GP passaged
A0867	#Lomuzio	GT 5	Puglia	goat	before 10 Nov 1984	ex IZS Puglia & Basilicata; GP passaged

Kudu ’93	is our standard comparison culture of GT 67 from Kudu in the Kruger National Park.