Q INSTITUT PASTEUR/ELSEVIER Paris 1992
Res. ViroL 1992, 143, 407-415
Rift Valley fever epizootic in the central highlands of Madagascar J. Morvan
(I)(*),
P.E. Rollin (2), S. Laventure (3), I. Rakotoarivony (3) and J. Roux (i)
a) Unit~ de Recherche sur les Arbovirus, Institut Pasteur, BP 1274, Antananarivo, (2) Laboratoire des Fi~vres hdmorragiques virales, lnstitut Pasteur, 75015 Paris, and (3) Unit~ d'Entomologie mddicale, Institut Pasteur de Madagascar
SUMMARY Between February and April 1991, unusual numbers of bovine abortion around Antananarivo (central highlands, Madagascar) were reported by official veterinary services. Rift Valley fever (RVF) virus isolations were made from sixteen aborted foetuses end one dead calf in different foci. Using monoclonal antibodies, the isolated viruses were found to be different from the 1979 RVF strains isolated in Madagascar from mosquitoes and human laboratory infection, and closer to African RVF strains. In a bovine population -- previously characterized by a negative or very low RVF antibody prevalence a high prevalence of IgM antibodies ( 2 6 4 / 9 9 4 : 2 6 . 5 % positive) was revealed; the IgM prevalence in recently aborting females varied from 40 to 9 1 % . Among 994 human sere tested by IgG-IFA (immunofluorescent antibody assay) and IgM ELISA, 8.2 % and 4.5 %, respectively, proved positive. A total of 11,371 mosquitoes ( 6 1 % Culex antennatus) were collected in the epizootic areas and tested without any virus isolation. Extensive studies were conducted to determine the geographical extension and the impact of this epidemic on the highly susceptible livestock and human populations. -
-
Key-words: RVF, Madagascar; Cattle, Man, Mosquito vectors, Antibodies, Virus isolation.
INTRODUCTION
Rift Valley fever (RVF) virus was first isolated from lambs in Kenya in 1930 (Daubney et al., 193 I). The viral infection was subsequently described in Sudan (Eisa and Obeid, 1977), Kenya (Davies, 1975), South Africa (Van Velden et el., 1977), Egypt (Meegan, 1979), Zimbabwe (Swanepoel, 1981) and Mauritania (Jouan et el., 1988). The presence of RVF in Madagas-
Submitted May 19, 1992, accepted October 26, 1992. (*) Correspondingauthor.
car was demonstrated in 1979 by the isolation of RVF virus from mosquitoes captured in a medium-altitude moist tropical forest (Clerc et al., 1982). In Madagascar, the disease was diagnosed in May 1990 among cattle at Fenerive, on the eastern coastal plain (Morvan et el., 1991a,b). In February 1991, numerous foci of bovine abortion were reported by the D~partement de Recherches zootechniques et v&~rinaires (DRZV) around Antananarivo in the central
408
.1. M O R V A N E T A L .
highlands (15 °/0 incidence of abortion among pregnant females). The diagnosis of RVF was rapidly established by virus isolation from an aborted foetus. We report here this new RVF epizootic in Madagascar, and present virological, serological and entomological data. The causes of this outbreak are discussed.
MATERIALS A N D METHODS Study area
The study was carried out in an area situated in the central highlands at an altitude of 1200-1400 m. The climate is temperate-tropical, the rainfall varies between 1,500 and 2,000 mm annually, with the heaviest rains from December to March, and with a dry season of 4 months (June-October). Mean temperature is 19°C (10-15°C in the coldest month). The central highlands constitute an area of grassy savanna and paddy-fields, with cattle bred for consumption. Herds are imported from cattle-producing regions (west, southwest) or Lake Alaotra in the north of the central highlands. Locations and abortion foci are presented in figure 1. M e t e o r o l o g i c a l statistics
Meteorological statistics were obtained from the Service National de M~t~orologie. They were calculated as follows: the mean rainfall for each calendar month was calculated for the 147-month period ; for each of the 147 values, the monthly mean was subtracted from the recorded value to give a positive or negative surplus. The surpluses for each month are given in figure 2.
Pasteur Institute staff from tail vein or jugular vein with a 10-ml vacutainer (Becton Dickinson, France). Sera were separated in the field by centrifugation and stored in liquid nitrogen. In addition, a serological investigation was conducted on farmers and families living around farms. Sera were stored at - 70°C before being tested. Mosquitoes were caught on human bait and with CDC light traps placed in cattle-pens between 18 h and 6 h. After species determination, monospecific pools were stored in liquid nitrogen.
Virus isolation and antigen d e t e c t i o n
Specimens (organ samples, cattle sera and mosquito pools) were inoculated into tissue culture (Vero E6 cells and Aedespseudoscutellaris cells) and intracranially into 1-2-day-old suckling mice according to standard techniques described by Digoutte et al. (1989). A standard indirect immunofluorescence test (IF) was used for identification in Vero cells using an RVF-virus-hyperimmune mouse ascitic fluid. Antigenic analysis was performed by IF using a panel of 8 monoclonal antibodies (kindly provided by J.F. Smith, Virology Division, USAMRIID, USA) directed against the glycoprotein G1 (4B4, 9B6), the glycoprotein G2 (3B9, IF6), the non-specific structural protein NSP31 (3C3, 3B4) and the nucleocapsid (R1P2E7, 9G3). The MgH 824 RVF virus strain isolated from man in Madagascar in 1979, and the E 501 Egyptian strain were used for antigenic comparison. The supernatant fluids, organs and mosquito pools were also tested for RVF virus antigen by an ELISA antigen detection method using an IgM trapping (Meegan et al., 1989; Morvan et al., 1991b). The identity of virus isolates was confirmed by complement fbcation (CF) at the WHO Collaborating Center for Reference and Research on Arboviruses (Pasteur Institute, Dakar).
Samples
Specimens for virus isolation (spleen, liver, brain and kidney of aborted foetus or dead animal) were collected by the DRZV between February and April in 7 farms where abortions were reported (fig. l). Specimens were sent to the Arbovirus Research Unit of the Institut Pasteur at Antananarivo. Bovine blood samples were collected by the
= Centers for Disease Control. = complement f'Lxation. DRZV = D~partement de Recherches zootechniques et
CDC CF
vc~t&inaires. ELISA = enzyme-linkedimmunosorbentassay.
Serological techniques
Sera were tested for IgG by an indirect immunofluorescent antibody assay (IFA) on slides coated with inactivated Vero E6 cells infected with RVF virus (ArB 1976) using labelled anti-human or antibovine immunoglobulin (Diagnostics' Pasteur,
IF IFA mAb RVF
= = = =
immunofluorescence(test). immunofluorescentantibodyassay. monoclonalantibody. Rift Valleyfever.
409
RIFT VALLEY FEVER EPIZOOTIC IN MADAGASCAR
lakeAOso~m
¢
Tslroanoma:dldy
PERINET
KIANJASOA e-~
TANJOMBAT~,ANTANANARIVO "~e Andram,slna .~
ANTANA
ANTSIRABE
N/RIVE • •~
City,Villages ComrnerciJcattle mlgrsUons VirusIsolations
Fig. 1. Map of Madagascar (with locations of serum sampling, virus isolations).
France). IgM-class antibodies were immunocaptured by an anti-I~ chain and detected by an ELISA technique (Digoutte eta/., 1989; Morvan et a/., 1991b): microplates were coated with goat anti-human or anti-bovine ~t-chain (Kirkegaard and Perry Laboratories, USA). The subsequent steps included successively: sera; RVF virus antigen (sucrose-acetoneextracted suckling mouse liver, Salk Institute, PA, USA); the detection system (RVF-virus-hyperimmune mouse ascitic fluid); the revealing system (goat anti-mouse globulin/horseradish peroxydase conjugate; Kirkegaard and Perry Lab.) and the substrate (O-tolidine, Sigma, USA).
26 bovine sera (aborted females) nor from 324 mosquito pools tested.
RESULTS
The IF test revealed a strongly positive reaction with RVF virus polyclonal antibody. The RVF virus antigen was detected in 23/40 (57.5 %) supernatant fluids by the ELISA method, and directly in the 24 corresponding specimens. The 17 isolates were confirmed by CF as RVF virus. The biological characteristics were similar for all isolates: a cytopathic effect was obtained 3-4 days after inoculation of Vero cells; the average survival time of suckling mice was 1-2 days post-inoculation.
Seventeen RVF virus strains were isolated from 40 (42.5 %) organs collected between February and April from different farms. Information concerning virus isolates is presented in table I. No RVF virus was isolated from
Antigenic analysis, using mAb, of the isolated strains is presented in table II. The 17 strains were recognized by all the monoclones but two (R1P2, 9B6), unlike the Madagascar strain isolated in 1979 from mosquitoes (MgH 824) and the Egyptian strain (E-501), which were recognized by the R1 P2 ati-nucleocapsid mAb.
Virus isolation
410
J. M O R V A N E T A L .
Table I. Results of RVF virus isolation and antigen detection during the 1991 RVF outbreak in the central highlands of Madagascar. Antigen Strain
Specimen
MgAn 990 MgAn 991 MgAn 992 MgAn 993 MgAn 994 MgAn 995 MgAn 996 MgAn 997 MgAn 998 MgAn 999 MgAn 1000 MgAn 1001 MgAn 1002 MgAn MgAn MgAn MgAn
1004 1005 1006 1007
AF AF AF AF AF AF AF AF AF AF AF AF AF AF CA AF AF AF AF AF AF AF AF CA
Organ liver spleen liver spleen brain liver spleen brain liver spleen liver spleen brain kidney pool lung liver spleen brain kidney liver spleen effusion spleen
Location Ivato 1 Ivato 1 Tanjombato Tanjombato Tanjombato Tanjombato Tanjombato Ivato 2 Ivato 2 Ivato 2 Tanjombato Tanjombato Tanjombato Tanjombato Tanjombato Kianjasoa Antsirabe 1 Antsirabe 1 Antsirabe 1 Antsirabe 1 Antsirabe 2 Antsirabe 2 Antsirabe 2 Antsirabe 2
Date
ELISA
Vero
28.01 28.01 28.01 28.01 06.02 06.02 06.02 06.02 06.02 06.02 07.02 07.02 07.02 07.02 21.01 10.03 27.03 27.03 27.03 27.03 22.04 22.04 22.04 23.04
+ + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + +
AF: aborted foetus; CA: calf.
Serology
Entomology
A total of 994 bovine sera were collected; 258/985 (26.2 07o)were found positive by IFA. Using ELISA IgM, 264/994 (26.5 07o) were found to be IgM-positive. The results of serological assays are given in table III. RVF virus antibodies were found in all sites, and the seroprevaience was significantly higher among aborting females (p < 10-6).
A total of 11,371 (324 pools) mosquitoes were collected (table V). Culex a n t e n n a t u s were predominant (more than 60 07o o f the collected mosquitoes) in the area around Antananarivo.
Among 994 human sera tested, 82/994 (8.2 070) and 45/994 (4.5 07o) were found positive by IFA and IgM-capture ELISA respectively (table IV). These data indicate lower RVF virus circulation in man.
The isolation o f 17 RVF virus strains from cattle and the high prevalence (26.5 %) o f IgM antibodies against RVF virus among bovine sera confirm the recent and intensive spread of the RVF epizootic among livestock in the central
DISCUSSION
RIFT VALLEY FEVER EPIZOOTIC IN MADAGASCAR
411
T a b l e I1. R e a c t i v i t y o f m A b b y i m m u n o f l u o r e s c e n t a s s a y w i t h t h e R V F v i r u s s t r a i n s i s o l a t e d d u r i n g t h e 1991 R V F o u t b r e a k in t h e c e n t r a l h i g h l a n d s o f M a d a g a s c a r . Anti-NC Strain
Anti-G 1
Anti-G2
R1P2
9G3
3C3
3B4
4B4
9B6
3B9
1F6
+ +
+ + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + +
_ (c) _ -
+ + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + +
E-501 (a) M g H 824 (b) M g A n 990 M g A n 991 M g A n 992 M g A n 993 M g A n 994 M g A n 995 M g A n 996 M g A n 997 M g A n 998 M g A n 999 M g A n 1000 M g A n 1001 M g A n 1002 M g A n 1004 M g A n 1005 M g A n 1006 M g A n 1007 (.) S t r a i n i s o l a t e d in E g y p t ;
Anti-NSP
-
-
-
-
c,) s t r a i n i s o l a t e d in 1 9 7 9 ( M a d a g a s c a r ) ;
co)
Res. ViroL 1992, 143, 407-415
Rift Valley fever epizootic in the central highlands of Madagascar J. Morvan
(I)(*),
P.E. Rollin (2), S. Laventure (3), I. Rakotoarivony (3) and J. Roux (i)
a) Unit~ de Recherche sur les Arbovirus, Institut Pasteur, BP 1274, Antananarivo, (2) Laboratoire des Fi~vres hdmorragiques virales, lnstitut Pasteur, 75015 Paris, and (3) Unit~ d'Entomologie mddicale, Institut Pasteur de Madagascar
SUMMARY Between February and April 1991, unusual numbers of bovine abortion around Antananarivo (central highlands, Madagascar) were reported by official veterinary services. Rift Valley fever (RVF) virus isolations were made from sixteen aborted foetuses end one dead calf in different foci. Using monoclonal antibodies, the isolated viruses were found to be different from the 1979 RVF strains isolated in Madagascar from mosquitoes and human laboratory infection, and closer to African RVF strains. In a bovine population -- previously characterized by a negative or very low RVF antibody prevalence a high prevalence of IgM antibodies ( 2 6 4 / 9 9 4 : 2 6 . 5 % positive) was revealed; the IgM prevalence in recently aborting females varied from 40 to 9 1 % . Among 994 human sere tested by IgG-IFA (immunofluorescent antibody assay) and IgM ELISA, 8.2 % and 4.5 %, respectively, proved positive. A total of 11,371 mosquitoes ( 6 1 % Culex antennatus) were collected in the epizootic areas and tested without any virus isolation. Extensive studies were conducted to determine the geographical extension and the impact of this epidemic on the highly susceptible livestock and human populations. -
-
Key-words: RVF, Madagascar; Cattle, Man, Mosquito vectors, Antibodies, Virus isolation.
INTRODUCTION
Rift Valley fever (RVF) virus was first isolated from lambs in Kenya in 1930 (Daubney et al., 193 I). The viral infection was subsequently described in Sudan (Eisa and Obeid, 1977), Kenya (Davies, 1975), South Africa (Van Velden et el., 1977), Egypt (Meegan, 1979), Zimbabwe (Swanepoel, 1981) and Mauritania (Jouan et el., 1988). The presence of RVF in Madagas-
Submitted May 19, 1992, accepted October 26, 1992. (*) Correspondingauthor.
car was demonstrated in 1979 by the isolation of RVF virus from mosquitoes captured in a medium-altitude moist tropical forest (Clerc et al., 1982). In Madagascar, the disease was diagnosed in May 1990 among cattle at Fenerive, on the eastern coastal plain (Morvan et el., 1991a,b). In February 1991, numerous foci of bovine abortion were reported by the D~partement de Recherches zootechniques et v&~rinaires (DRZV) around Antananarivo in the central
408
.1. M O R V A N E T A L .
highlands (15 °/0 incidence of abortion among pregnant females). The diagnosis of RVF was rapidly established by virus isolation from an aborted foetus. We report here this new RVF epizootic in Madagascar, and present virological, serological and entomological data. The causes of this outbreak are discussed.
MATERIALS A N D METHODS Study area
The study was carried out in an area situated in the central highlands at an altitude of 1200-1400 m. The climate is temperate-tropical, the rainfall varies between 1,500 and 2,000 mm annually, with the heaviest rains from December to March, and with a dry season of 4 months (June-October). Mean temperature is 19°C (10-15°C in the coldest month). The central highlands constitute an area of grassy savanna and paddy-fields, with cattle bred for consumption. Herds are imported from cattle-producing regions (west, southwest) or Lake Alaotra in the north of the central highlands. Locations and abortion foci are presented in figure 1. M e t e o r o l o g i c a l statistics
Meteorological statistics were obtained from the Service National de M~t~orologie. They were calculated as follows: the mean rainfall for each calendar month was calculated for the 147-month period ; for each of the 147 values, the monthly mean was subtracted from the recorded value to give a positive or negative surplus. The surpluses for each month are given in figure 2.
Pasteur Institute staff from tail vein or jugular vein with a 10-ml vacutainer (Becton Dickinson, France). Sera were separated in the field by centrifugation and stored in liquid nitrogen. In addition, a serological investigation was conducted on farmers and families living around farms. Sera were stored at - 70°C before being tested. Mosquitoes were caught on human bait and with CDC light traps placed in cattle-pens between 18 h and 6 h. After species determination, monospecific pools were stored in liquid nitrogen.
Virus isolation and antigen d e t e c t i o n
Specimens (organ samples, cattle sera and mosquito pools) were inoculated into tissue culture (Vero E6 cells and Aedespseudoscutellaris cells) and intracranially into 1-2-day-old suckling mice according to standard techniques described by Digoutte et al. (1989). A standard indirect immunofluorescence test (IF) was used for identification in Vero cells using an RVF-virus-hyperimmune mouse ascitic fluid. Antigenic analysis was performed by IF using a panel of 8 monoclonal antibodies (kindly provided by J.F. Smith, Virology Division, USAMRIID, USA) directed against the glycoprotein G1 (4B4, 9B6), the glycoprotein G2 (3B9, IF6), the non-specific structural protein NSP31 (3C3, 3B4) and the nucleocapsid (R1P2E7, 9G3). The MgH 824 RVF virus strain isolated from man in Madagascar in 1979, and the E 501 Egyptian strain were used for antigenic comparison. The supernatant fluids, organs and mosquito pools were also tested for RVF virus antigen by an ELISA antigen detection method using an IgM trapping (Meegan et al., 1989; Morvan et al., 1991b). The identity of virus isolates was confirmed by complement fbcation (CF) at the WHO Collaborating Center for Reference and Research on Arboviruses (Pasteur Institute, Dakar).
Samples
Specimens for virus isolation (spleen, liver, brain and kidney of aborted foetus or dead animal) were collected by the DRZV between February and April in 7 farms where abortions were reported (fig. l). Specimens were sent to the Arbovirus Research Unit of the Institut Pasteur at Antananarivo. Bovine blood samples were collected by the
= Centers for Disease Control. = complement f'Lxation. DRZV = D~partement de Recherches zootechniques et
CDC CF
vc~t&inaires. ELISA = enzyme-linkedimmunosorbentassay.
Serological techniques
Sera were tested for IgG by an indirect immunofluorescent antibody assay (IFA) on slides coated with inactivated Vero E6 cells infected with RVF virus (ArB 1976) using labelled anti-human or antibovine immunoglobulin (Diagnostics' Pasteur,
IF IFA mAb RVF
= = = =
immunofluorescence(test). immunofluorescentantibodyassay. monoclonalantibody. Rift Valleyfever.
409
RIFT VALLEY FEVER EPIZOOTIC IN MADAGASCAR
lakeAOso~m
¢
Tslroanoma:dldy
PERINET
KIANJASOA e-~
TANJOMBAT~,ANTANANARIVO "~e Andram,slna .~
ANTANA
ANTSIRABE
N/RIVE • •~
City,Villages ComrnerciJcattle mlgrsUons VirusIsolations
Fig. 1. Map of Madagascar (with locations of serum sampling, virus isolations).
France). IgM-class antibodies were immunocaptured by an anti-I~ chain and detected by an ELISA technique (Digoutte eta/., 1989; Morvan et a/., 1991b): microplates were coated with goat anti-human or anti-bovine ~t-chain (Kirkegaard and Perry Laboratories, USA). The subsequent steps included successively: sera; RVF virus antigen (sucrose-acetoneextracted suckling mouse liver, Salk Institute, PA, USA); the detection system (RVF-virus-hyperimmune mouse ascitic fluid); the revealing system (goat anti-mouse globulin/horseradish peroxydase conjugate; Kirkegaard and Perry Lab.) and the substrate (O-tolidine, Sigma, USA).
26 bovine sera (aborted females) nor from 324 mosquito pools tested.
RESULTS
The IF test revealed a strongly positive reaction with RVF virus polyclonal antibody. The RVF virus antigen was detected in 23/40 (57.5 %) supernatant fluids by the ELISA method, and directly in the 24 corresponding specimens. The 17 isolates were confirmed by CF as RVF virus. The biological characteristics were similar for all isolates: a cytopathic effect was obtained 3-4 days after inoculation of Vero cells; the average survival time of suckling mice was 1-2 days post-inoculation.
Seventeen RVF virus strains were isolated from 40 (42.5 %) organs collected between February and April from different farms. Information concerning virus isolates is presented in table I. No RVF virus was isolated from
Antigenic analysis, using mAb, of the isolated strains is presented in table II. The 17 strains were recognized by all the monoclones but two (R1P2, 9B6), unlike the Madagascar strain isolated in 1979 from mosquitoes (MgH 824) and the Egyptian strain (E-501), which were recognized by the R1 P2 ati-nucleocapsid mAb.
Virus isolation
410
J. M O R V A N E T A L .
Table I. Results of RVF virus isolation and antigen detection during the 1991 RVF outbreak in the central highlands of Madagascar. Antigen Strain
Specimen
MgAn 990 MgAn 991 MgAn 992 MgAn 993 MgAn 994 MgAn 995 MgAn 996 MgAn 997 MgAn 998 MgAn 999 MgAn 1000 MgAn 1001 MgAn 1002 MgAn MgAn MgAn MgAn
1004 1005 1006 1007
AF AF AF AF AF AF AF AF AF AF AF AF AF AF CA AF AF AF AF AF AF AF AF CA
Organ liver spleen liver spleen brain liver spleen brain liver spleen liver spleen brain kidney pool lung liver spleen brain kidney liver spleen effusion spleen
Location Ivato 1 Ivato 1 Tanjombato Tanjombato Tanjombato Tanjombato Tanjombato Ivato 2 Ivato 2 Ivato 2 Tanjombato Tanjombato Tanjombato Tanjombato Tanjombato Kianjasoa Antsirabe 1 Antsirabe 1 Antsirabe 1 Antsirabe 1 Antsirabe 2 Antsirabe 2 Antsirabe 2 Antsirabe 2
Date
ELISA
Vero
28.01 28.01 28.01 28.01 06.02 06.02 06.02 06.02 06.02 06.02 07.02 07.02 07.02 07.02 21.01 10.03 27.03 27.03 27.03 27.03 22.04 22.04 22.04 23.04
+ + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + +
AF: aborted foetus; CA: calf.
Serology
Entomology
A total of 994 bovine sera were collected; 258/985 (26.2 07o)were found positive by IFA. Using ELISA IgM, 264/994 (26.5 07o) were found to be IgM-positive. The results of serological assays are given in table III. RVF virus antibodies were found in all sites, and the seroprevaience was significantly higher among aborting females (p < 10-6).
A total of 11,371 (324 pools) mosquitoes were collected (table V). Culex a n t e n n a t u s were predominant (more than 60 07o o f the collected mosquitoes) in the area around Antananarivo.
Among 994 human sera tested, 82/994 (8.2 070) and 45/994 (4.5 07o) were found positive by IFA and IgM-capture ELISA respectively (table IV). These data indicate lower RVF virus circulation in man.
The isolation o f 17 RVF virus strains from cattle and the high prevalence (26.5 %) o f IgM antibodies against RVF virus among bovine sera confirm the recent and intensive spread of the RVF epizootic among livestock in the central
DISCUSSION
RIFT VALLEY FEVER EPIZOOTIC IN MADAGASCAR
411
T a b l e I1. R e a c t i v i t y o f m A b b y i m m u n o f l u o r e s c e n t a s s a y w i t h t h e R V F v i r u s s t r a i n s i s o l a t e d d u r i n g t h e 1991 R V F o u t b r e a k in t h e c e n t r a l h i g h l a n d s o f M a d a g a s c a r . Anti-NC Strain
Anti-G 1
Anti-G2
R1P2
9G3
3C3
3B4
4B4
9B6
3B9
1F6
+ +
+ + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + +
_ (c) _ -
+ + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + +
E-501 (a) M g H 824 (b) M g A n 990 M g A n 991 M g A n 992 M g A n 993 M g A n 994 M g A n 995 M g A n 996 M g A n 997 M g A n 998 M g A n 999 M g A n 1000 M g A n 1001 M g A n 1002 M g A n 1004 M g A n 1005 M g A n 1006 M g A n 1007 (.) S t r a i n i s o l a t e d in E g y p t ;
Anti-NSP
-
-
-
-
c,) s t r a i n i s o l a t e d in 1 9 7 9 ( M a d a g a s c a r ) ;
co)
