TRANSACTIONSOFTHE ROYAL. SOCIETYOFTROFICAL MEDICINE AND HYGIENE (1992) 86,263-265

Low seroprevalence

of Toxoplasma

go&ii

antibodies

263

in a Tanzanian

village

E. Gillel A. Bj6rkman3, I. Rooth2,3, I. Ljungstriim’ and E. Linder’ ‘Department of Parasitology, National Bacteriological Laboratory, Stockholm, Sweden; 2Malaria Research Unit, Nyamisati, Tanzania; 3Department of Infectious Diseases, Roslagstull Hospital, Karolinska Institute, Stockholm, Sweden Abstract

The prevalence of antibodies against Toxoplasma gondii was studied in the population of Nyamisati village in Tanzania using the direct agglutination test, indirect immunofluorescence test, and immunosorbent agglutination test. All positive sera were positive by both direct agglutination and indirect immunofluorescence tests and were confirmed by the dye test. The seropositivity was confirmed by immunoblotting showing a distinct 32 kDa band in all the seropositive samples. The seropositivity rate was 4% (191450)among the subjects of Nyamisati origin and 47% (15132)among immigrants from other areasof Tanzania. Most of the infections appeared to have occurred between 5 and 15 years of age. The generally low transmission in this mainly Muslim village appeared to be related to sparse consumption of contaminated food and low prevalence of oocystsdue to scarcity of felines. Introduction Toxoplasma gondii is a cosmopolitan protozoan para-

site. The systemic infection remains latent in the body after the acute stage. During the acute stage there is a strong, specific antibody response. Antibodies persist lifelong at a lower level. Toxoplasmosis is transmitted to humans mainly by the ingestion of tissue cysts present in undercooked pork or mutton. However, in the developing world, intake of oocysts from soil or, occasionally, water contaminated by cats or other felines is also important and in some areas the main route of infection (FRENKEL& RUIZ, 1981; BENENSON~~ al., 1982). From different parts of Tanzania there have been reports of 77% and 40% seroprevalence in the general population (BENNETT et al., 1970; ROEVER-BONNET, 1972) and 42% among healthy pregnant women (GILL &

The presenceof IgM class anti-Toxoplasma antibodies at a dilution 1:50 was determined by a modification of the immunosorbent agglutination assay (DESMONTSet al., 1981) with all samples possessing IgG class antibodies, and the dye-test (SABIN & FELDMAN, 1948) was performed on all positive samples and on a corresponding number of randomly selectednegative sera. Immunoblotting was used to confirm the results of serological routine assays.T. gondii extracts prepared by incubation for 30 min in 1% Nonidet NP 40@in phosphate-buffered saline were separatedby sodium dodecyl sulphate-polyacrylamide gel electrophoresis (LAEMMLI, 1970), using a 10% continuous slab gel. Transfer of proteins to nitrocellulose membranes(Bio-Rad Laboratories, USA) was performed by a modification of the technique described by TOWBINet al. (1979).

MTIMAVALYE, 1982).

The aim of this study was to determine the seroprevalences obtained with different serological methods including immunoblotting in a Tanzanian village where neither pork nor mutton was eaten and where cats were not kept as pets. Materials and Methods Study area and population

Nyamisati is an isolated rural community surrounded by mangrove swamps in the delta of the Rufijii river 140 km south of Dar es Salaam, Tanzania. The inhabitants are predominantly Muslims. A few immigrants to the village are Christians. The main diet is rice and fish, on rare occasionschicken or meat but never pork or mutton. Cats are never kept as pets and very few stray cats exist in the surrounding area. No other felines are known to be present in the area. Sample collection

During 1988 venous blood samples were collected by venepuncture from all 482 individuals over 2 years of age in the village. Sera were separated after sedimentation and stored at - 18°Cfor a few months in the village before transportation to Sweden, where they were stored at 40°C. Before examination, the serawere heat-inactivated for 30 min at 56°C. Serological assays

All sera were examined for immunoglobulin (Ig) G anti-Toxoplasma antibodies by means of a direct agglutination test (DESMONTS & REMINGTON, 1980)performed in microtitre plates (ToxoScreen DA@ BioMerieux, France). Titres 2 1:40 were regarded aspositive. Positive sera were further examined by an indirect immunofluorescence test using acetone-fixed T. gondii RH strain trophozoites as antigen (HULDT et al., 1975). Author for correspondence: Ewert Linder, National BacteriologicalLaboratory,Departmentof Parasitology,S-10521 Stockholm, Sweden.

Results

Of the 482 samplesexamined, 34 (7%) had detectable IgG class anti-Toxoplasma antibodies. IgM class antiToxoplasma antibodies were not detected in any of the 34 seropositive cases.All the results of direct agglutination Table 1. Toxoplasma gondii seropositivity in the population of Nyamisati village, Tanzania, according to geographical origin and religion

Nyamisati residents Muslims

Religion 450 Total no. examined 19 (4%) No. positive

Immigrants to Nyamisati Muslims Christians 9 (ii%,

6 ($0/b)

tests were in agreementwith those of the dye test and indirect immunofluorescence test. The positive IgG titres ranged from 1:40 to 1:32 000 in the agglutination assay, from 1:10 to 1:810 in the indirect immunofluorescence assay,and from 1:lO to 1:6250in the dye test. The seropositive serum samples reacted with several different antigenic components by immunoblotting. All positive sera reacted with a band in the 32 kDa region, and most of them also with a band in the 25 kDa region. Table 2. The relation of Toxoplasmagondii seropositivitywith agein Nyamisati village, Tanzania

Agegroup(years) 2-5 Total no. examined 37 0 No. positive

615

1630

>30

8 (&)

10 $6,)

,6$%)

All ages 3:&

Someof the seronegative sera also showed slight reactivity in the 32 kDa region, but the reaction was faint and irregular (Figure).

264

kDa 4 94 4 67

4 -30 4 20.1 4 14.4

A. Seropositive kDa 4 94 4 67 4 43

B. Seronegative Figure.Westernblotsof ToxopZasma gomfii antigenextraction.The nitrocellulosestripscontainingthe antigensweretreatedwith serumof T. go&ii seropositive(A) andseronegative (B) subjects.The seropositivesera(A) all showedreactivityin the 32 kDa regionandmostof themalsoiq the 25 kDa region. The seronegative sera(B) showedonlyafaint,irregularpattern.

The sera with IgG anti-Toxoplasma antibodies were obtained from 20 males and 14 females. Personswho had been born in Nyamisati had a seropositivity rate of 4% (19/450), whereas the immigrants had a rate of 47% (G/32) (Table 1). Twelve of the seropositivt persons belonged to the samefamilies (parent-child, husband-wife, brother-sister-husband). The relation of seropositivity to age is shown in Table 2. The main increase in seropositivity appearedto occur in the agegroup 6-15 years. Discussion

The observed 7% overall seroprevalencerate contrasts with the previously reported high prevalences of toxoplasmosis in Tanzania (BENNETT et al., 1970; ROEVERBONNET, 1972; GILL & MTIMAVALYE, 1982). The fact that the different tests used gave consistent results indicates that the observed low seroprevalencewas real. The results also indicate the usefulness of immunoblotting as a confirmatory test for toxoplasmosis.

The diet in the Nyamisati area, mainly rice and fish, does not favour transmission of Toxoplasma tissue cysts. Furthermore, food is generally well cooked, in contrast to the area in northern Tanzania inhabited by the hunting Hadza tribe (77% seropositive) who consume meat partly raw (BENNETT et al., 1970). In populations with early seroconversion, contaminated soil has been considered the main route of transmission. The fact that we found no infection among children below the ageof 6 years therefore indicates little transmission occurring by oocysts. Because of the low number of definitive feline hosts in this rural area, the load of oocysts in the soil was probably low in spite of favourable climatic conditions. The transmission of toxoplasmosis appears to vary significantly between different areas of Tanzania. Travelling may therefore expose individuals from ‘non endemic’ areasto a higher risk of acquiring infections and may thus pose a risk for susceptible pregnant women of

265

infection of the foetus and consequent adverseeffects. Acknowledgements

We gratefully thank Britt Andersson, Barbro Fahlgren, Maria Karpinska and Madeleine Rafto for technical assistance. The study received support from the Swedish Pentecostal Church. A. Bjorkman is holder of a grant from the Swedish Medical ResearchCouncil. References

Benenson, M. W., Takafuji, E. T., Lemon, S. M., Greenup, R. L. & Sulzer, A. J. (1982). Oocysts-transmitted toxoplasmosis associated with ingestion of contaminated water. New EnglandJournal of Medicine, 307,666-669.

Bennett, F. J., Kagan, I. G., Barnicot, N. A. & Woodburn, J. C. (1970). Helminth and protozoa1parasites of the Hadza of Tanzania. Transactions of the Royal Society of Tropical Medicine and Hygiene, 64,857-880.

Desmonts, G. & Remington, J. S. (1980). Direct agglutination test for diagnosis of Toxoplasma infection: method for increasing sensitivity and specificity. Journal of Clinical Microbiology, 11,562-568.

Desmonts, G., Naot, Y. & Remington, J. S. (1981). An IgM immunosorbent agglutination assayfor diagnosis of infectious diseases: diagnosis of acute congenital and acquired Toxoplasma infection. Journal of Clinical Microbiology, 14, 486491.

Frenkel, J. K. & Ruiz, A. (1981). Endemicity of toxoplasmosis in Costa Rica. Transmission between cats. soil. intermediate hosts and humans. American Journal of Epidemiology, 113, 254-269. Gill, H. S. & Mtimavalye, L. A. R. (1982). Prevalence of Toxoplasma antibodies in pregnant African women in Tanzania. AfricanJournal

ofMedicine andMedicalSciences,

11, 167-170.

Huldt, G., Ljungstriim, I. & Aust-Kettis, A. (1975). Detection by immunofluorescence of antibodies to parasitic agents. Use of class-specific conjugates. Annals of the New York Academy of Sciences, 254,3OC314.

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685. Roever-Bonnet, H. (1972). Toxoplasmosis in tropical Africa. Tropical and Geographical Medicine, 24,7-13.

Sabin, A. B. & Feldman, H. (1948). Dyes as microchemical indicators of a new immunity phenomenon affecting a protozoon parasite (Toxoplasma). Science, 108,660-663. Towbin, H., Staehelin, T. & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Science of the USA, 76,4350-4354. Received 21 August accepted for publication

1990; revised 8 November 13 November 1991

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The 12th Seminar on Amebiasis will be held on 12-14 November 1992 at the National Academy of Medicine, Mexico City. Free communications are invited on all aspectsof basic and clinical research on Entamoeba histolytica and related Entamoeba. For further details contact Dr Adolf0 Martinez-Palomo, CINVESTAV-IPN, Apartado Postal 1C740,070OOMexico, D.F., Mexico, Fax (525) 754-5116,or Dr Louis S. Diamond, Bldg 4, Rm 126, National Institutes of Health, Bethesda, MD 20892, USA, Fax (301)-496-2443.

1991;

Low seroprevalence of Toxoplasma gondii antibodies in a Tanzanian village.

The prevalence of antibodies against Toxoplasma gondii was studied in the population of Nyamisati village in Tanzania using the direct agglutination t...
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