Meir Yoeli

A genetic investigation of virulence in a rodent malaria parasite

The genetic basis of virulence in a line (YM) of Plasmodium yoelii yoelii was investigated in a cross with a mild line (A/C). The blood forms of the virulent line developed extensively in mature erythrocytes of mice, causing death of the host within 7 days; infections with the mild line were normally restricted to reticulocytes, infected animals recovering after three weeks. Lines YM and A/C differed additionally in enzyme and drug-sensitivity markers. Studies on infections established from each line alone from sporozoite mixtures of the two lines and from the cross between the lines showed that the appearance of virulence had been caused by a genetic change in the parasite, and not by other factors such as a concurrent infection with another organism. An analysis of the characters of 56 clones derived from the cross showed that the virulence character had undergone recombination with the other markers, and appeared to be inherited in Mendelian fashion. Three clones exhibited atypical virulence, although it was not clear whether this had been produced by genetic recombination.

Effects of temperature on sporogonic development of Plasmodium berghei.

The effect of temperature on the sporogonic cycle of Plasmodium berghei was studied in two experimental mosquito vectors. Complete sporogonic development takes place in Anopheles quadrimaculatus at 16 to 21 C, and in A. stephensi at 16 to 24 C. The upper temperature limits for development appear to be the lowest reported for any plasmodia. The sporogonic development of P. berghei is apparently adapted to the narrow temperature range which is found in the microclimate of its natural vector. High temperatures have a more rapidly damaging effect on the early than on the late stages of sporogony. Mosquitoes exposed to 28 C for as short a time as 4 hr following their infective blood meal fail to develop sporozoites. If this high temperature treatment is withheld until the parasite completes the early stages of sporogonic development at a favorable temperature, exposure to at least 3 days of 28 C is necessary to induce visible damage. The mode of action of high temperature damage is unknown at present. Ever since the discovery of the rodent malarial parasite, Plasmodium berghei, by Vincke and Lips (1948), laboratory investigations involving the sporogonic stages of development of the parasite have been impeded by the inability to find a suitable experimental vector. Recent work (Yoeli, Most, and Bone, 1964; Vanderberg and Yoeli, 1965) has shown that the parasite can be readily transmitted by several species of anophelines if the infected mosquitoes are maintained at a temperature of about 21 C. This is the approximate daytime temperature that the natural vector, Anopheles dureni, is subjected to in its microclimate within the forest galleries of the Congo during the transmission season. Previous experiments conducted in our laboratory in which mosquitoes were kept at 28 C only rarely resulted in successful cyclical development. P. berghei, apparently, is unusual among the malarial parasites in that its sporogonic stages develop optimally at relatively low temperatures. After our initial success at cyclical transmission of P. berghei by experimentally infected mosquitoes, we thought it useful to examine in more critical detail the effects that Received for publication 1 December 1965. *This work, contribution number 36 from the Army Research Program on Malaria, was carried out under the sponsorship of the Commission on Malaria, Armed Forces Epidemiological Board, and supported in part by the U. S. Office of the Surgeon General, Department of the Army, and by research grant AI-02423 from the NIAID, NIH, U. S. Public Health Service. temperature has on the entire sporogonic cycle

Parasitic infections in a closed community. Results of a 10-year survey in Willowbrook State School.

Abstract A study designed to elucidate the causes of mass morbidity and widespread intestinal disorders in Willowbrook State School has revealed the role of parasitic agents in the aetiology of the diseases. Strongyloides stercoralis, Hymenolepis nana and Giardia lamblia were the predominant pathogenic agents. Their spread was limited, however, to the childrens buildings and to the severely mentally retarded. 522 patients with S. stercoralis and 322 with H. nana were detected during the period 1962–1970. Annual stool surveys served to trace symptomless carriers, to determine levels of endemicity and to assess the effects of treatment and control. Factors which influence the spread of parasitic infections are discussed in the light of findings of the survey and preventive measures for custodial institutions are outlined.

The biology of a newly isolated strain of Plasmodium berghei in a rodent host and in experimental mosquito vectors.

Abstract 1. 1) A strain of Plasmodium berghei , Kasapa S.P. 7307, recently isolated from the Belgian Congo, has been cyclically transmitted through Anopheles quadrimaculatus, Anopheles aztecus , and young white rats three successive times. Sporozoites from crushed oocysts of heavily infected mosquitoes were used as inoculum. 2. 2) The newly isolated “passage” strains were designated as N.Y.U. 1 , N.Y.U. 2 , and N.Y.U. 3 Strains of P. berghei (New York University Strains). 3. 3) The pre-patent period in four sporozoite infected young rats were: 71 hours, 96 hours, 8 days, and 10 days. 4. 4) Parasites found in the peripheral blood at the early “flooding” period of 71 hours after sporozoite inoculation are described in detail. 5. 5) A description is given of the different developmental stages of the sporozonic cycle as observed in the newly isolated strains, under experimental conditions.

PLASMODIUM BERGHEI: CYCLICAL TRANSMISSIONS BY EXPERIMENTALLY INFECTED ANOPHELES QUADRIMACULATUS.

A number of strains of Plasmodium berghei were isolated from sporozoites of Anopheles dureni. Laboratory-bred Anopheles quadrimaculatus fed on carriers of the newly isolated strains showed overwhelming midgut infections and moderate or mild salivary gland infections. Successive cyclic transmissions by the bite of experimentally infected A. quadrimaculatus in laboratorybred tree rats (Thamnomys surdaster) were carried out.

Plasmodium berghei ookinete formation in vitro.

Abstract Blood containing gametocytes of Plasmodium berghei (N.K. 65 strains) taken up by Anopheles stephensi during the infective blood meal and rapidly ejected during the act of engorgement induced formation of large numbers of ookinetes in vitro when the blood was subsequently kept in heparinized capillary tubes for 22–24 hours at 21 °C. A parallelism between ookinete formation in vitro and oocyst development in vivo has been observed in these studies. Aqueous extracts of A. stephensi midguts added to blood containing gametocytes of P. berghei induced fertilization and ookinete formation of P. berghei under similar experimental conditions. Extracts from blood obtained from engorged A. stephensi failed to induce any marked ookinete formation in vitro . Clumping and close adherence of ookinetes has been observed in vitro . Tightly clustered groups of macrogametocytes and macrogametes were noted both in the in vitro blood stained smears and also in some midgut preparations from infected A. stephensi , 22–24 hours after the infective blood meal. It is assumed that ookinete agglutination originates in earlier adherence and clumping of the fertilized gametes.

Altered Dihydrofolate Reductase Associated with Drug-Resistance Transfer between Rodent Plasmodia

Resistance to pyrimethamine in strains of Plasmodium vinckei and of Plasmodium berghei is attributed to changes in amounts and properties of the dihydrofolate reductases. The resistant strain of Plasmodium berghei was isolated from an experimentally induced mixed infection of drug-resistant Plasmodium vinckei and drug-sensitive Plasmodium berghei, through biological filtration in hamsters. The drug resistance in Plasmodium berghei appears to have been acquired through transfer of part of the gene or genes coding for dihydrofolate reductase from the resistant Plasmodium vinckei to the sensitive Plasmodium berghei.

Drug-resistance transfer among rodent plasmodia. 1. Acquisition of resistance to pyrimethamine by a drug-sensitive strain of Plasmodium berghei in the course of its concomitant development with a pyrimethamine-resistant P. vinckei strain.

A study on transfer of drug resistance between species of malaria parasites was carried out in mixed infections of P. berghei and P. vinckei . The biological characters which served as contrasting markers for separation of the two species were the inability of the ‘old’ blood-passaged strain of P. vinckei to develop in suitable mosquito vectors and its failure to produce parasitaemia in the adult golden hamster. Facility of sporogonic development and cyclical transmission and a pronounced virulence for the adult hamster marked the N.K. 65 strain of P. berghei . Development of a maximal and stable resistance to 200 mg/kg pyrimethamine in the P. vinckei strain was achieved by a gradual increase in drug level in successive blood transfers. Mixed, simultaneous infections of pyrimethamine-resistant P. vinckei and sensitive P. berghei were experimentally induced in white mice. Subinoculations of the mixed infection at the height of parasitaemia was carried out in adult hamsters. Suppression of the P. vinckei infection in the hamster by innate resistance and the reappearance of P. berghei in the blood was noted. Tests with the maximal concentration of pyrimethamine (200 mg/kg) showed the acquisition of drug resistance in some of the P. berghei infected hamsters. Persistence of the acquired resistance was demonstrated following fifteen successive blood transfers in the absence of drug pressure and after five cyclical transmissions. Parallel control experiments with pyrimethamine-sensitive P. vinckei and P. berghei in mixed infections failed to reveal any enhancement in drug resistance. No spontaneous drug-resistant mutant was found in the sensitive P. berghei after exposure to a single course of treatment with the drug, and persistence of parasites in some treated animals was considered as individual variations in host reactions to the drug. This resistance never persisted after blood transfer or cyclical transmission. No loss in drug resistance had been found following transfer of the resistant R strain from mouse to hamster. A marked difference in the course of the plasmodial infection and in the length of survival of mice inoculated with the drug-resistant P. berghei was noted following treatment with pyrimethamine in various concentrations. It is suggested that these differences represent a selective action of the drug on a heterogenic P. berghei population, consisting of drug-resistant and drug-sensitive parasites in the blood of the host. Injection of sonified material from pyrimethamine-resistant P. vinckei and P. berghei into mice inoculated with a drug-sensitive P. berghei failed to induce enhancement of resistance to the drug. The phenomenon of drug-resistance transfer observed under experimental conditions in rodent malaria is viewed in the light of similar phenomena in micro-organisms. We gratefully acknowledge the assistance rendered by Mr Jerold Sklarsh. This paper is Contribution no. N 408 from the Army Research Programme on Malaria. The work was carried out under the sponsorship of the Commission on Malaria, Armed Forces Epidemiological Board, and supported in part by the United States Office of the Surgeon General, Department of the Army.

Plasmodium berghei: Mechanisms and sites of resistance to sporogonic development in different mosquitoes☆

Abstract An experimental study of the mechanisms and patterns of resistance to Plasmodium berghei in different mosquito species revealed a diversity of factors which prevent or inhibit sporogonic development in its different phases and in different sites in the mosquito vector. The experiments showed that Culex salinarius was a totally resistant species in which exflagellation and ookinete formation are prevented. In Aedes aegypti, ookinetes in small or moderate numbers are formed but penetration of the mosquito midgut wall is blocked and oocysts are not formed. In the three experimental vectors, Anopheles quadrimaculatus, Anopheles aztecus, and Anopheles stephensi grades of enhanced susceptibility are recognized. They are expressed in lesser numbers of abnormal and degenerative oocysts, in higher numbers of sporozoites in the salivary gland, and greater viability and invasiveness of these sporozoites. In Anopheles dureni, the natural vector of rodent malaria, one observes both in nature and under experimental conditions the highest adaptation and most pronounced grade of susceptibility to P. berghei.

Studies on filariasis. III. Partial growth of the mammalian stages of Dirofilaria immitis in vitro

Abstract Invasive third-stage larvae of Dirofilaria immitis obtained from experimentally infected Anopheles quadrimaculatus were maintained in a culture medium in a perfusion apparatus for 30 days at 37 °C. Molting of the larvae was observed, and an increase in size from an initial length of 1059 μ and a 27.68 μ width to an average 1510 μ length and 34.70 μ width at 12 days, and a 1825 μ length and 38.70 μ width at 30 days, were recorded. Blunting and rounding of the anterior ends in the growing larvae were conspicuous, and the caudal papillae were prominent. These changes are comparable to those occurring in larvae of similar age found in experimentally infected dogs. The results of the experiments are discussed and compared in the light of previous attempts to grow D. immitis in vitro.