Genfrequenzen und Vererbung im Duffy-System

Summary Genotyping in the Duffy-system was carried out using special combined trypsin-antiglobulin-techniques. The heterozygous presence of the third allele Fy was estimated by the single dose of the antigen Fy^a or Fy^b, whereas the homozygous types Fy^aFy^a or Fy^bFy^b gave the double-dosage-effect.Genotyping on 1408 unrelated persons in Hesse gave the following gene frequencies: Fy^a=0.4251; Fy^b=0.5582; Fy=0.0167. Expected and observed values showed a good correspondence (p0.72 for 4 d.f.).In phenotyping of 3768 persons, the very rare type Fy (a-b-) was found only once, which is in good agreement with the calculated gene frequency of Fy.Among those genotyped, family studies were subsequently done on 22 selected propositi. In the 13 cases in which the propositus had the third allele in the heterozygous state, the same third allele was again found at least once in the family where the propositus was a child (7x). Where the propositus was a parent (6x), the third allele was also found three times in the children; this allele was not transmitted in only 3 families with 4 children. In contrast, we did not find a single Fy-gene in the 8 families where the propositus was Fy^aFy^a or Fy^bFy^b.Finally, phenotyping was done in 283 unselected families. In this group, genotyping was restricted to those cases in which the distribution of Duffy-phenotypes suggested the presence of the third allele. In both groups of families not a single case of contradiction against the hereditary rules was observed.The reliability of genotyping is discussed, as well as its importance for anthropological studies and paternity cases.

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Direktor: Profl Dr. W. Spielmann

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Vaccination with Plasmodium knowlesi AMA1 formulated in the novel adjuvant co-vaccine HT™ protects against blood-stage challenge in rhesus macaques

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a leading blood stage vaccine candidate. Plasmodium knowlesi AMA1 (PkAMA1) was produced and purified using similar methodology as for clinical grade PfAMA1 yielding a pure, conformational intact protein. Combined with the adjuvant CoVaccine HT™, PkAMA1 was found to be highly immunogenic in rabbits and the efficacy of the PkAMA1 was subsequently tested in a rhesus macaque blood-stage challenge model. Six rhesus monkeys were vaccinated with PkAMA1 and a control group of 6 were vaccinated with PfAMA1. A total of 50 µg AMA1 was administered intramuscularly three times at 4 week intervals. One of six rhesus monkeys vaccinated with PkAMA1 was able to control parasitaemia, upon blood stage challenge with P. knowlesi H-strain. Four out of the remaining five showed a delay in parasite onset that correlated with functional antibody titres. In the PfAMA1 vaccinated control group, five out of six animals had to be treated with antimalarials 8 days after challenge; one animal did not become patent during the challenge period. Following a rest period, animals were boosted and challenged again. Four of the six rhesus monkeys vaccinated with PkAMA1 were able to control the parasitaemia, one had a delayed onset of parasitaemia and one animal was not protected, while all control animals required treatment. To confirm that the control of parasitaemia was AMA1-related, animals were allowed to recover, boosted and re-challenged with P. knowlesi Nuri strain. All control animals had to be treated with antimalarials by day 8, while five out of six PkAMA1 vaccinated animals were able to control parasitaemia. This study shows that: i) Yeast-expressed PkAMA1 can protect against blood stage challenge; ii) Functional antibody levels as measured by GIA correlated inversely with the day of onset and iii) GIA IC₅₀ values correlated with estimated in vivo growth rates.     

Determination of the Duffy genic frequencies in Senegal

Duffy gene frequencies, determined in Senegal, are as follows: Fy^a=0,0171−Fy^b=0,057−Fy=0,9772. A comparison made with European and African results confirms the prevalence of the Fy gene in Black populations. Such a study never was realized in Senegal.     

Circulating immune complexes in rodent and simian malaria

The circulating immune complexes have been detected in the sera of albino rats infected withPlasmodium berghei and rhesus monkeys infected with P.knowlesi by (i) quantitative cryoprecipitation assay and (ii) polyethylene glycol assay. In the rodent model, the levels of circulating immune complexes increased during infection and decreased considerably in the post-infection period. In the simian system, high levels were detected during peak parasitaemia. Polyethylene glycol precipitate obtained from the sera during acuteP. knowlesi infection when analysed by Immunoelectrophoresis was found to contain (i) monkey IgG, (ii) four other components of monkey plasma, (iii) two components of normal monkey erythrocytes and (iv) antigen(s) ofP. knowlesi.     

Application of a multiplex PCR genotyping assay of 31 red blood cell antigens for establishment of a panel of reference DNA in China

DNA based blood group genotyping has been widely used in clinical blood transfusions, and a number of different molecular blood group testing methods have been developed, including the detection of single nucleotide polymorphism (SNP) and genomic DNA sequencing. As the molecular bases of blood groups can differ widely between ethnic groups, a set of reference DNAs, especially for the Chinese population, is required for the development and validation of these methods, and for their optimal use in routine practice in China. In this study, a total of 100 DNA samples obtained from 60 established cell lines and 40 Chinese blood donors were typed for 31 red blood cell antigens of 13 blood group systems using a multiplex polymerase chain reaction (PCR) assay. Finally, nine DNA samples were selected to establish a panel of reference DNA that included M, N, S, s, Mur, Lu^a, Lu^b, Au^a, Au^b, K, k, Fy^a, Fy^b, Jk^a, Jk^b, Di^a, Di^b, Sc1, Sc2, Do^a, Do^b, Co^a, Co^b, Kn^a, Kn^b, In^b, Vel antigens and Fy (a-b-) null phenotype.     

Cross-reactivity in rapid diagnostic tests between human malaria and zoonotic simian malaria parasite Plasmodium knowlesi infections

Plasmodium knowlesi has a relatively broad host range extending to humans, in whom it causes zoonotic malaria. Recent studies have shown that human infection with P. knowlesi is widely distributed in forested areas of Southeast Asia. In the present study, we evaluated commercial rapid diagnostic tests (RDTs) for human malaria to assess their reactivity and sensitivity in detecting P. knowlesi parasites using blood samples obtained from infected monkeys. The blood samples were assayed using two commercial RDTs based on immunochromatographic assays: (i) the OptiMAL-IT, designed to detect parasite lactate dehydrogenase (pLDH) of both P. falciparum and other plasmodia, and (ii) the Entebe Malaria Cassette (MC), designed to detect P. falciparum-specific histidine-rich protein 2 (PfHRP2) and P. vivax-specific pLDH. Interestingly, when the P. knowlesi-infected blood samples were examined with the RDTs, OptiMAL test results were interpreted as falciparum malaria-positive, while Entebe MC test results were interpreted as vivax malaria-positive. The sensitivities of both tests in detecting P. knowlesi parasite were similar to those for P. falciparum and higher than P. vivax. Thus, commercial RDTs based on detection of pLDH should be used with great caution, and should not replace conventional microscopy in the diagnosis of suspected cases of P. knowlesi malaria.     

Blutgruppen und Lepra bei moçambiquanischen Völkerschaften

Zusammenfassung Etwa 600 moçambiquanische Eingeborene, vorwiegend Chuabo und Macua wurden auf folgende Blutgruppensysteme bzw. Merkmale untersucht: A B 0, M N S s S^u, C C^wc D E e, K k Kp^aJs^aJs^b, P₁, Fy^aFy^bFy, Jk^aJk^b, Le^a, Di^a, Gc, SEPh, PGM₁, PGM₂ und ADA.Im Durchschnitt gesehen überwiegen die typischen Negermerkmale bei den Moçambiquanern mehr als bei anderen negriden Populationen. Signifikante Unterschiede zwischen verschiedenen Stämmen, insbesondere zwischen Macua, Chuabo, Bitonga und Changane, wurden nicht gefunden. In nahezu allen Systemen unterschieden sich dagegen die leprösen von den nichtleprösen Macua mehr oder weniger deutlich. Im AB0-, MNSS^us-, Rhesus-, Lewis-, Gc- und PGM-System sind die Unterschiede sogar signifikant. Zur Zeit haben wir keine Erklärung für diese Befunde.

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Blood groups and lepra in populations of moçambique

Summary About 600 natives of Moçambique, preferably Chuabo and Macua were tested for the following blood group systems, resp. markers: A B 0, M N S s S^u, C C^wc D E e, K k Kp^aJs^aJs^b, P₁, Fa^aFy^bFy, Jk^aJk^b, Le^a, Di^a, Gc, SEPh, PGM₁, PGM₂ and ADA.The typical blood group markers for negroes were found to a higher extent than in nearly all the other negroid populations. Significant differences between the single tribes of Moçambique, especially between Macua, Chuabo, Bitonga and Changane were not found. In almost all systems, however, marked differences between leprous and non-leprous Macuas could be detected. These were statistically significant in the AB0-, Rhesus-, Lewis-, Gc- and P.GM-system. At this time no explanation for these findings can be given.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Ultrastructural Study of Plasmodium knowlesi Antigen Used in Vaccination of Rhesus Monkeys*

SYNOPSIS. Material from various steps obtained in the French pressure cell technic of preparing antigen from Plasmodium knowlesi-infected red cells, was examined by electron microscopy. A positively charged colloidal iron solution was used to differentiate between membranes of host red cells and parasites. Red cell membranes take the stain, whereas parasite membranes do not. This antigen which has been used previously to protect monkeys against P. knowlesi appears to consist almost entirely of membrane-bounded vesicles. Some of these vesicles contain a fine granular material, whereas others appear empty. The antigen failed to stain with the positively charged iron solution, which suggests that it is free of contamination by host cell membrane.     

Blood groups and types,hemoglobin variants,and G-6-PD deficiency among Abu Dhabians in the United Arab Emirates

Some erythrocyte genetic factors were studied in the indigenous population of Abu Dhabi, the capital of the United Arab Emirates, on the south-eastern coast of the Arabian peninsula. Determinations carried out included blood groups and types ABO, MNS, Rh_o, KkJs^a, Fy^aFy^b, P₁, Le^a, Vel^a, hemoglobin variants, and screening for G-6-PD deficiency. Prevalence of most blood groups and types harmonized with that among neighboring Arabs and some Arabs elsewhere. The MS and NS gene complexes were noticeably high. African admixture was expressed by the presence of Js^a and Hb S and large numbers of Fy. G-6-PD deficiency was rather high.     

Purine and pyrimidine nucleotide profiles during synchronous malaria infection (Plasmodium knowlesi) in the rhesus monkey

Webster H. K., Haut M. J., Martin L. K. and Hildebrandt P. K. 1982. Purine and pyrimidine nucleotide profiles during synchronous malaria infection (Plasmodium knowlesi in the rhesus monkey. International Journal for Parasitology12: 75–79. Blood levels of purine and pyrimidine nucleotides were determined during synchronous infection by Plasmodium knowlesi in rhesus monkeys. Infected monkeys followed over 2–3 intraerythrocytic cycles showed variations in nucleotide pool levels characteristic of the predominant schizogonic growth stage. These changes in nucleotide levels as described for ‘ring’-stage, trophozoite growth and schizogony indicate a cyclically varying relationship between nucleotide concentrations and a specific stage of parasite development during the blood-phase of malaria infection.     

Zur formalen Genetik des Duffy-Systems

The formal genetics of the Duffy-polymorphism was examined, using reagents Anti-Fy(a) and Anti-Fy(b). Our family series comprises 247 unselected families from the southwestern part of Germany with 459 children; extramarital children are excluded from this investigation. The results agree with the hypothesis 3 alleles Fy^a, Fy^b, Fy at an autosomal locus.

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Direktor: Prof. Dr. Dr. H. Baitsoh

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Human-type blood groups and Gm factors in gibbons (Hylobates)

Blood specimens from 69 gibbons (63Hylobates lar, 4Hylobates concolor, and 2Hylobates pileatus) were tested for human-type ABO, MN, and Rh blood groups. AmongH. lar, three phenotypes were noted in the ABO and MN blood groups respectively, but all fourH. concolor were grouped as AM. All group A gibbons were of subgroup A₁; subgroups A₂B and A₁₂B were observed at a low frequency in group AB gibbons. Le^b antigen was detected in about 30% of the red cell samples fromH. lar, but all the samples were negative for Le^a. All the gibbons tested had c(hr) antigen but no other Rh antigens (D, C, E, and e) in their red cells. Some selected blood samples fromH. lar were also tested for some other blood group antigens and for the Gm and Inv factors. The Jk^a antigen was detected in all the red cell samples tested, but the S, s, U, K, k, and Fy^a antigens were not. In the tests of plasma with anti-Gm (1),H. lar could be divided into two groups, i.e., Gm(1)^Gi and Gm(–1)^Gi; Gm(2), Gm(4), and Inv(1) were absent in the species.     

Sequence,evolution and ligand binding properties of mammalian Duffy antigen/receptor for chemokines

The Duffy antigen/receptor for chemokine, DARC, acts as a widely expressed promiscuous chemokine receptor and as the erythrocyte receptor for Plasmodium vivax. To gain insight into the evolution and structure/function relations of DARC, we analyzed the binding of anti-human Fy monoclonal antibodies (mAbs) and human chemokines to red blood cells (RBCs) from 11 nonhuman primates and two nonprimate mammals, and we elucidated the structures of the DARC genes from gorilla, gibbon, baboon, marmoset, tamarin, night monkey and cattle. CXCL-8 and CCL-5 chemokine binding analysis indicated that the promiscuous binding profile characteristic of DARC is conserved across species. Among three mAbs that detected the Fy6 epitope by flow cytometric analysis of human and chimpanzee RBCs, only one reacted with night monkey and squirrel monkey. Only chimpanzee RBCs bound a significant amount of the anti-Fy3 mAb. Fy3 was also poorly detected on RBCs from gorilla, baboon and rhesus monkey, but not from new world monkeys. Alignment of DARC homologous sequences allowed us to construct a phylogenetic tree in which all branchings were in accordance with current knowledge of primate phylogeny. Although DARC was expected to be under strong internal and external selection pressure, in order to maintain chemokine binding and avoid Plasmodium vivax binding, respectively, our present study did not provide arguments in favor of a selection pressure on the extracellular domains involved in ligand specificity. The amino acid variability of DARC-like polypeptides was found to be well correlated with the hydrophylicity indexes, with the highest divergence on the amino-terminal extracellular domain. Analysis of the deduced amino acid sequences highlighted the conservation of some amino acid residues, which should prove to be critical for the structural and functional properties of DARC.     

In vivo study of human Plasmodium knowlesi inMacaca fascicularis

Plasmodium knowlesi is a malaria parasite of Old World monkeys and is infectious to humans. In this study Macaca fascicularis was used as a model to understand the host response to P. knowlesi using parasitological and haematological parameters. Three M. fascicularis of either sex were experimentally infected with P. knowlesi erythrocytic parasites from humans. The pre-patent period for P. knowlesi infection in M. fascicularis ranged from seven to 14 days. The parasitemia observed was 13,686-24,202 parasites per μL of blood for asexual stage and 88-264 parasites per μL of blood for sexual stage. Periodicity analysis adopted from microfilaria periodicity technique of asexual stage showed that the parasitemia peak at 17:39 h while the sexual stage peaked at 02:36 h. Mathematical analysis of the data indicates that P. knowlesi gametocytes tend to display periodicity with a peak (24:00-06:00) that coincides with the peak biting activity (19:00-06:00) of the local vector, Anopheles latens. The morphology of P. knowlesi resembled P. falciparum in early trophozoite and P. malariae in late trophozoite. However, it may be distinguishable by observing the appliqué appearance of the cytoplasm and the chromatin lying inside the ring. Haematological analysis on macaques with knowlesi malaria showed clinical manifestations of hypoglycaemia, anaemia and hyperbilirubinemia. Gross examination of spleen and liver showed malaria pigments deposition in both organs.     

An electron cytochemical study of 6-phosphogluconate dehydrogenase activity in infected erythrocytes during malaria

The distribution of 6-phosphogluconate dehydrogenase (6-PGD) was examined by an electron microscopic technique in erythrocytes of rodents infected with $\text{Plasmodium berghei}$, chickens with $\text{P. gallinaceum}$ and rhesus monkeys with $\text{P. knowlesi}$. Unlike glucose-6-phosphate dehydrogenase, which is restricted to the host erythrocyte, 6-PGD was found to be present in the parasite as well as the host erythrocyte in all infections studied. The implications of these findings are discussed in relation to the metabolism of malaria parasites.     

Plasmodium knowlesi: In vitro biosynthesis of methionine

Methionine biosynthesis was studied in rhesus monkey erythrocytes infected with Plasmodium knowlesi malaria which were cultured in vitro with l-[3-¹⁴C]serine, methyl-[¹⁴C]tetrahydrofolic acid, and l-[³⁵S]homocysteine. Radioactivity derived from [3-¹⁴C]serine was detected in approximately equivalent amounts in methionine and thymidylic acid by thin-layer chromatography of acid-hydrolysates of washed erythrocytes. The results with methyl-[¹⁴C]tetrahydrofolic acid were inconclusive. Radioactivity from l-[³⁵S]homocysteine also appeared in methionine but the level of homocysteine required for maximal activity was tenfold that of serine. The results indicate that the serine: 5,10-methylenetetrahydrofolic acid: 5-methyl-tetrahydrofolic acid: methionine biosynthetic pathway is present in the P. knowlesi malaria parasite.     

Genotyping of the Duffy Blood Group among Plasmodium knowlesi-Infected Patients in Malaysia

The Duffy blood group is of major interest in clinical medicine as it plays an important role in Plasmodium knowlesi and Plasmodium vivax infection. In the present study, the distribution of Duffy blood group genotypes and allelic frequencies among P. knowlesi infected patients as well as healthy individuals in Peninsular Malaysia were determined. The blood group of 60 healthy blood donors and 51 P. knowlesi malaria patients were genotyped using allele specific polymerase chain reaction (ASP-PCR). The data was analyzed using Fisher''s exact test in order to assess the significance of the variables. Our results show a high proportion of the FY*A/FY*A genotype (>85% for both groups) and a high frequency of the FY*A allele (>90% for both groups). The FY*A/FY*A genotype was the most predominant genotype in both infected and healthy blood samples. The genotype frequency did not differ significantly between the donor blood and the malaria patient groups. Also, there was no significant correlation between susceptibility to P. knowlesi infection with any Duffy blood genotype.     

Conservation note: Socioecology and conservation of macaques and langurs in Varanasi,India

A survey of rhesus monkeys (Macaca mulatta) and hanuman langurs (Presbytis entellus) was conducted in urban and forest areas of Varanasi, India. Data were collected on group size and composition. Nine groups of rhesus monkeys inhabited various urban niches, but langurs were completely absent from urban areas. Approximately 159 rhesus monkeys and 720 langurs lived in the forested area. Both the urban and the forested habitats of this area are being reduced in size, and it is suggested that these populations be moved to nearby forest preserves.