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MEMORANDUM FOR Defense Intelligence Agency, ATTN: DT-5C

Filename:0pgv0014.oop



AFMIC-S&TI (340d)

Subject: MEMORANDUM FOR Defense Intelligence Agency, ATTN: DT-5C
[   (b)(6)   ], Washington, D.C. 20340-6053

SUBJECT: AFMIC Assessment Based on Laboratory Analysis of Iraqi
Serum Samples by U.S. Army Medical Research Institute for
Infectious Diseases (USAMRIID) 

1.        The 71 samples in question were obtained during 
Operation Desert Storm. No medical history presently is available 
on the individuals from whom the blood was collected. (See 
enclosure 1).

2.        Only a portion of the specimens were screened for 
botulinum toxin neutralizing antibody. Methods used to screen the 
group are contained in enclosure 1.

3.        Results of analysis of the samples for immunization 
against anthrax and botulinum toxin, possible Iraqi BW agents:

a. Anthrax--According to the attached USAMRIID report, during 
initial serological screens, four sera of 69 were found to be 
seropositive for Bacillus anthracis reactive antibody using the 
enzyme immune assay (EIA). The EIA reactivity was confirmed by 
western blot analysis. The sera contained reactivity against 
protective antigen but not lethal factor by western blot analysis. 
This suggests that the serum donors have been exposed to B. 
anthracis antigens by natural infections or vaccinations. Having 
proper histories (background, occupation, etc.) would help to 
assess possible sources of natural exposure).

       AFMIC cannot assess the immunization status of all Iraqi 
soldiers against anthrax based on the information obtained from 
exploitation of these samples alone. Because we do not know the 
character of the Iraqi vaccine and because the kinetics of the 
decay of antibodies post-vaccination for that vaccine is not 
known, it is impossible to say unequivocally whether the antibody 
titer is a result of active disease or immunization. However, it 
can be stated that 1) not all Iraqi military are immunized against 
biological warfare agents and 2) certain sectors of the Iraqi 
military may receive immunization against anthrax.

b.        Botulinum toxin-- A total of 7 samples were tested for 
anti-botulinum toxin activity. [   (b)(1) sec 1.3(a)(4)   ]




 None of the 7 samples tested were found to contain botulinum 
toxin neutralizing antibody at or above the 0.04 IU level.

      None of the donor individuals whose sera were tested appear 
to have been immunized against botulinum toxin. Since the 
populatlon sample was small, no judgement can be made as to 
whether the Iraqis are immunizing any of their troops against 
botulinum toxin.

4.       [   (b)(1) sec 1.3(a)(4)   ]















5.        Positive reactions obtained from sera testing for 
Coxiella burnetti, Vaccinia virus, Sandfly fever virus, West Nile 
virus, Rift Valley fever (RVF) virus, and Sindbis virus conform to 
plausible expectations resulting from natural exposure to endemic 
disease agents, routine vaccinations, or non-specific cross 
reactions.  The results from testing for these endemic diseases 
has significantly increased the confidence level of AFMIC's 
infectious desease threat assessment.

6. (U) AFMIC POC [   (b)(6)   ], [   (b)(2)   ]

[   (b)(6)   ]

[   (b)(2)   ]








United States Army Medical Institute for Infectious Diseases, 
Special Pathogens Section (SPS), Department of Epidemiology, 
Disease Assessment Division

SPECIMEN NUMBERS: [   (b)(2)   ]

SPECIMEN DESCRIPTION. USAMRIID received, on 31 January 1991, 71 
samples [   (b)(2)   ]
                          collected during Operation Desert Storm.

ANALYSIS PERFORMED. [   (b)(1) sec 1.3(a)(4)   ]


only a portion of the specimens were screened for botulinum toxin 
neutralizing antibody. Since the chance of finding a specimen 
seronegative for botulinum toxin serotype A neutralizing antibody 
but seropositive for botulinum toxin serotype B, E or F antibody 
is considered to be unlikely, the following procedure was used to 
screen the 31 January survey group. First, a random 10% sample of 
the sera was tested for neutralizing antibody against serotype A 
botulinum toxin at a level of 0.04 IU. This level of antibody is 
easily attained in man through immunization with a reasonably good 
toxoid. Second, any sample found to be seropositive for serotype A 
botulinum toxin neutralizing antibody was rescreened for serotype 
B, E, and F reactivity at the 0.04 IU level. Third, any specimen 
not in the 10% sample and found to be seropositive for B. 
anthracis reacting antibody was tested for botulinum toxin 
neutralizing antibody following the protocal described for the 10% 
sample.

During the initial serological screens, four sera, [   (b)(2)   ]

 were found to be seropositive for B. anthracis reactive antibody 
using the enzyme immune assay (EIA); the EIA reactivity was 
confirmed by western blot analysis. Three sera, [   (b)(2)   ]

were screened for botulinum toxin serotype A activity at a level 
of 0.04 IU based upon these findings. Because of the small amount 
of serum, the fourth sample [   (b)(2)   ]                  was 
depleted during the first series of test and could not be screened 
for anti-botulinum toxin activity.

RESULTS. Serum samples [   (b)(2)   ]

 were found to be seronegative for botulirlum toxin serotype A 
neutralizing activity and are considered not to contain botulinum 
toxin neutralizing antibody at or above the 0.04 IU level.



[   (b)(6)   ]




United States Army Medical Institute for Infectious Diseases, 
Special Pathogens Section (SPS), Department of Epidemiology, 
Disease Assessment Division

SPECIMEN NUMBERS: [   (b)(2)   ]

SPECIMEN DESCRIPTION: USAMRIID received 71 serum samples on 31 
January 1991. The samples were received in plastic freezer vials 
wrapped in ziplock bags. The vials contained 0.3 to 3 mls and were 
numbered [   (b)(1) sec 1.3(a)(4)   ]



ANALYSIS PERFORMED: The samples were screened for evidence of 
exposures or vaccinations against: botulinum toxin, B. anthracis, 
F. tularensis, Y. Pestis, C. burnetii, vaccinia virus Crimean-
Congo hemorrhagic Fever virus, Rift Valley fever virus, sindbis 
virus, West Nile virus, sandfly fever virus (Naples and Sicilian 
strains), dengue virus, and Venezuelan equine encephalitis virus.

RESULTS SUMMARY: The results are presented in Table 1. None of 
the serum specimens tested reacted with antigens from Cl. 
botulinum, Y Pestis, F tularensis, CCHF virus, dengue virus type 2 
or Venezuelan equine encephalitis virus. Varying numbers of sera 
reacted with antigens from the remaining agents: 4/69 reacted with 
B. anthracis, 7/69 reacted with C. burnetii, 8/69 reacted with 
vaccinia virus, 1/69 reacted with Rift Valley fever virus, 18/69 
reacted with West Nile virus 26/69 reacted with Sandfly virus 
(Naples strain), and 55/69 reacted with Sandfly virus (Sicilian 
strain).  

RECOMMENDATIONS: [   (b)(1) sec 1.3(a)(4)   ]







[   (b)(6)   ]







RESULTS: Specimens [   (b)(2)   ]


TABLE 1 TEST RESULTS SUMMARY ON 31 JANUARY SHIPMENT.

								Serological Screen (a)	
	Agent 				Test (b)			No.Pos/Total 
(l)

B. anthracis				EIA	   			4/69 (2)
Cl. botulinum toxin A		NEUT	    			0/7 (3)
C. burnetii				EIA	   			7/69 (4) 
Y. Pestis					EIA	   			0/69
F. tularensis				MIA	   			0/69
Vaccinia virus				IFA	   			8/69 (5)
CCHF virus				EIA	   			0/69
RVF virus					EIA	   			1/69
Sindbis virus				EIA	   			3/69 (6)
West Nile virus			EIA	  			18/69 (7)
Sandfly fever virus			EIA	  			26/69 (8)
 (Naples strain)
Sandfly fever virus			EIA	  			55/69 (8)
 (Sicilian strain)
Dengue virus Type 2			EIA	   			0/69
VEE virus					EIA	   			0/69
(a) Numbers in parenthesis pertain to paragraphs in the Result 
Interpretation section of the report. 
(b) Abbreviations: EIA, enzyme immune assay; IFA, 
immunofluorescent antibody assay; and MIA, microagglutination 
test.





RESULTS INTERPRETATION: [   (b)(2)   ]


1. Sample size. [   (b)(6)   ][   (b)(2)   ]









The vial numbers are listed in Table 2 along with corresponding 
sample identification numbers from the CBW SAMPLE DOCUMENTATION 
form.  [   (b)(2)   ]








[   (b)(1) sec 1.3(a)(4)   ][   (b)(2)   ]







2. Bacillus anthracis reactivity. Four specimens were positive for 
B. anthracis antibody by EIA. The sera [   (b)(2)   ]

 contained reactivity against protective antigen but not lethal 
factor by western blot analysis. The results suggest the serum 
donors have been exposed to B. anthracis antigens by natural 
infections or vaccinations. Consideration should be given to 
conducting follow-up interviews of serum [   (b)(2)   ]

donors; [   (b)(1) sec 1.3(a)(4)   ]

history associated with the acquisition of B. anthracis reactive 
antibody maybe important.

3. Botulinum toxin reactivity. [   (b)(2)   ]                     
 

        only a portion of the 69 specimens were screened for 
botulinum toxin neutralizing antibody. Since the chance of finding 
a specimen seronegative for botulinum toxin serotype A 
neutralizing antibody but seropositive for botulinum toxin 
serotype B, E or F antibody is considered to be unlikely, the 
following procedure was followed to screen the 31 January survey 
group. First, a random 10% sample of the nonprioritized sera was 
tested for neutralizing antibody against serotype A botulinum 
toxin at a level of 0.04 IU. This level of antibody is easily 
attained in man through immunization with a reasonably good 
toxoid. Second, any sample found to be seropositive for serotype A 
botulinum toxin neutralizing antibody was rescreened for serotype 
B, E, and F reactivity at the 0.04 IU level. Third, any specimen 
not in the lO% sample and found to be seropositive for B. 
anthracis reacting antibody was tested for botulinum toxin 
neutralizing antibody using the protocol described for the lO% 
sample.

Seven sera were tested for botulinum toxin serotype A antibody and 
found to be seronegative. 3 of the 4 [   (b)(2)   ]

           sera found to be seropositive for B. anthracis reactive 
antibody are presently being screened for botullinum toxin 
serotype A activity. Because of the small amount of serum, the 
fourth sample [   (b)(2)   ]                  was depleted during 
the first series of test and can not be screened for anti-
botulinum toxin activity.

4. Coxiella burnetii reactivity. Seven sera [   (b)(2)   ]


 were positive for C. burnetti antigen reactivity in the EIA 
suggesting the serum donors had been infected in the past.

5. Vaccinia virus reactivity. Eight sera [   (b)(2)   ]



reacted with vaccinia virus antigens in the IFA test. The 
reactions were low titered; the titers were lower than that seen 
in sera from U.S. troops serosurveyed two months after being 
vaccinated against smallpox.

6. Sindbis Virus reactivity. Three sera [   (b)(2)   ]

 reacted with sindbis virus antigens in the EIA. The 
seroreactivity was not unexpected since sindbis virus is endemic 
in the Middle East and natural infections may be common in Iraq.

7. West Nile virus antigen reacivity Eighteen sera reacted with 
West Nile virus (WNV) antigens in the EIA. The seroreactivity was 
not unexpected since WNV is endemic in the Middle East and natural 
infections may be common in Iraq. The WNV seroreactive specimens 
are listed in Table 4.

8. Sandfly fever virus antigen reactivity. Twenty six sera reacted 
with antigens from the Naples strain of sandfly fever virus and 
fifty five sera reacted with antigens from the Sicilian strain of 
sandfly fever virus. The seroreactivity was not unexpected since 
these viruses are endemic in the Middle East and natural 
infections may be common in Iraq. The results of the Sandfly fever 
virus serology is prssented in Table 4.




TABLE 2. 31 JANUARY 1991 SHIPMENT SPECIMEN DOCUMENTATION.

SAMPLE NUMBER	IDENTIFICATION NUMBER (1)		VIAL NUMBER (2)

[   (b)(2)   ]



TABLE 3 ISOENZYME LEVELS IN SPECIMENS WITH IDENTICAL NUMBERS

SAMPLE NUMBERS							CPK 		DONORS
IDENTIFICATION NUMBER (l)	VIAL N0.(2) 	LEVEL(3)	IDENTITY NO.

[   (b)(2)   ]



TABLE 4. SPECIMENS REACTING WITH WEST NILE VIRUS (WNV). SANDFLY 
FEVER, VIRUS NAPLES (SFVN) AND SANDFLY FEVER VIRUS SICILIAN (SFVS) 
ANTIGENS.

SAMPLE IDENTIFICATION NUMBER					VIRUSES
								WNV 		SFVN 	SFSV

[   (b)(2)   ]						-		+		+
[   (b)(2)   ]						-		-		+
[   (b)(2)   ]						-		+		+
[   (b)(2)   ]						-		-		+
[   (b)(2)   ]						-		-		+
[   (b)(2)   ]						-		-		+
[   (b)(2)   ]						+		+		+
[   (b)(2)   ]						-		+		+
[   (b)(2)   ]						-		-		+
[   (b)(2)   ]						-		+		+
[   (b)(2)   ]						+		+		+
[   (b)(2)   ]						+		-		+
[   (b)(2)   ]						+		+		+
[   (b)(2)   ]						-		+		+
[   (b)(2)   ]						-		-		+
[   (b)(2)   ]						+		+		+
[   (b)(2)   ]						-		-		-
[   (b)(2)   ]						-		-		+
[   (b)(2)   ]						-		-		+
[   (b)(2)   ]						-		-		+
[   (b)(2)   ]						-		-		+
[   (b)(2)   ]						+		-		+
[   (b)(2)   ]						-		-		+
[   (b)(2)   ]						+		+		+
[   (b)(2)   ]						-		-		+
[   (b)(2)   ]						-		-		+
[   (b)(2)   ]						+		+		+
[   (b)(2)   ]						+		+		+
[   (b)(2)   ]						-		-		+
[   (b)(2)   ]						+		+		+
[   (b)(2)   ]						+		+		+
[   (b)(2)   ]						-		-		+
[   (b)(2)   ]						+		+		+
[   (b)(2)   ]						+		+		+
[   (b)(2)   ]						+		-		+
[   (b)(2)   ]						-		-		+
[   (b)(2)   ]						-		+		+
[   (b)(2)   ]						+		-		+
[   (b)(2)   ]						-		+		+
[   (b)(2)   ]						-		+		+
[   (b)(2)   ]						-		-		+
[   (b)(2)   ]						-		+		-
[   (b)(2)   ]						-		-		+
[   (b)(2)   ]						-		+		-

(1). Symbols: -, negative reaction; +, positive reaction.



conti.

TABLE 4. SPECIMENS REACTING WITH WEST NILE VIRUS (WNV), SANDFLY 
FEVER VIRUS NAPLES (SFVN), AND SANDFLY FEVER VIRUS SICILIAN (SFVS) 
ANTIGENS.



SAMPLE IDENTIFICATION NUMBER					VIRUSES
								WNV 		SFVN 	SFVS


[   (b)(2)   ] 					-		+		+
[   (b)(2)   ] 					-		-		+
[   (b)(2)   ] 					+		-		+
[   (b)(2)   ] 					-		-		+
[   (b)(2)   ] 					-		-		+
[   (b)(2)   ] 					-		-		+
[   (b)(2)   ] 					+		-		+
[   (b)(2)   ] 					-		-		+
[   (b)(2)   ] 					+		-		+
[   (b)(2)   ] 					-		+		+
[   (b)(2)   ] 					-		+		+
[   (b)(2)   ] 					-		-		+
[   (b)(2)   ] 					-		+		+
[   (b)(2)   ] 					-		+		+

(1). Symbols: -, negative reaction; +, positive reaction

 



 

 



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