Peter G. Contacos

Studies on the Transmission of Simian Malarias. I. Transmission of Two Strains of Plasmodium inui by Anopheles maculatus and A. stephensi.

Five species of Anopheles mosquitoes were examined in regard to their susceptibility to infection with the OS and L strains of Plasmodium inui. Anopheles maculatus and A. stephensi were readily infected and were able to transmit each of the strains by bite. Three New World mosquitoes, A. quadrirraculatus, A. freeborni, and A. albimanus, became infected. The A. quadrimaculatus rarely exhibited sporozoite-positive salivary glands. The other two species failed to produce gland infections. The malaria parasite, Plasmodium inui, naturally infects monkeys throughout much of South and Southeast Asia. This parasite has a quartan periodicity and has been isolated from Macaca irus, M. mulatta, M. nemestrina, M. radiata, and Cynopithecus niger (see Eyles, 1963, for a discussion of this parasite and its geographical and host distribution). The development of this species to the sporozoite level in mosquitoes has been demonstrated in A. atroparvus (Weyer, 1937; Garnham, 1951; Mohiuddin, 1957; and Shortt et al., 1963), in A. quadrimaculatus (see Eyles, 1960) and in A. stephensi (see Shortt et al., 1963). Garnham (1951) obtained transmission of this species to M. mullatta (rhesus monkey) by the bites of infected A. atroparvus mosquitoes coupled with the intravenous inoculation of dissected salivary glands. Mohiuddin (1957) passaged the parasite to Al. miulatta by the inoculation of dissected glands from infected A. aztecus. He also fed infected A. atroparvus and A. aztecus on a monkey which later developed a patent infection. In this latter instance, it is impossible to say which species of mosquito transmitted the parasite. Eyles (1960) obtained transfer by the intravenous inoculation of dissected salivary glands from infected A. quadrimaculatus. Shortt et al. (1963) transferred P. inui by the intravenous inoculation of salivary glands containing sporozoites from A. aztecus and from A. stephensi. The only transmission by mosquito bite appears to be that reported above by Mohiuddin (1957). A number of strains of P. inui are being investigated in this laboratory. Reported here Received for publication 4 April 1966. 664 are the results of mosquito infection and transmission studies involving two different strains of P. inui malaria and five species of Anopheles mosquitoes. MATERIALS AND METHODS The strains of malaria used were the OS and L strains of P. inui. For information dealing with the origin and designation of the OS strain see Coatney et al. (1966). The L strain of P. inui was isolated from a naturally infected A. leucosphyrus mosquito collected in Malaya by Wharton et al. (1962). This parasite, like the OS strain, was obtained from our laboratory in Malaysia. The A. quadrimaculatus mosquitoes were the Q-1 strain which was obtained from Technical Development Laboratories, CDC, Savannah, Georgia, and maintained in our laboratory since 1959. The A. freeborni (F-1 strain) were from Marysville, California, and have been maintained in the laboratory since 1944. The A. stephensi were from Delhi, India, and were obtained from the London School of Hygiene and Tropical MNedicine, London, England in 1963. The A. albimanus mosquitoes were the A-9 strain originally obtained from San Salvador, and maintained in our laboratory since 1960. The A. maculatus were obtained from the Institute for Medical Research, Kuala Lumpur, Malaysia, in 1964, and have been maintained in our laboratory since then. Monkeys (M. mulatta) were infected by the intravenous inoculation of parasitized blood or by intrahepatic inoculation of sporozoites. When gametocytes were present in the peripheral blood, mosquitoes were allowed to feed. All mosquitoes were insectary reared and after feeding were incubated at a temperature of 78 to 80 F and at high humidity. Mosquitoes were fed 5% Karo syrup daily on a cotton pledget. Dissections to determine presence and number of oocysts on the gut were begun 6 days after feeding. Since transmission was the primary goal of these studies, only a minimum number of mosquito guts were examined (normally five to 11). Salivary gland dissections were made only after This content downloaded from 157.55.39.185 on Thu, 26 May 2016 04:40:27 UTC All use subject to http://about.jstor.org/terms COLLINS ET AL.-TRANSMISSION OF PLASMODIUM INUI BY ANOPHELES SPP. 665 TABLE I. Infection of Anopheles maculatus mosquitoes with the OS strain of Plasmodium inui. Days postGametocytes per inoculation 100 WBC Mosquitoes Per cent Avg no. Monkey (Days of dissected infection oocysts per patency) Male Female pos. gut Blood-induced infections T-163 7 20 44 7 100 38 8 50 60 6 100 46 9 20 260 5 100 37 T-166 8 4 18 8 75 42

The Chesson strain of Plasmodium vivax in Aotus monkeys and anopheline mosquitoes.

The Chesson strain of Plasmodium vivax was studied in Aotus trivirgatus monkeys. Parasitemia in intact and splenectomized animals was similar to that reported for this strain in man. Comparative infectivity studies with mosquitoes fed on infected monkeys indicated that the most susceptible was Anopheles freeborni, followed by An. balabacensis, An. culicifacies, An. maculatus, An. atroparvus, An. stephensi, An. quadrimaculatus, and An. albimanus. Transmissions via sporozoites from An. maculatus was demonstrated on two occasions; prepatent periods were 30 and 32 days.

Transmission of four central American strains of Plasmodium vivax from monkey to man.

Infections of 4 different Central American strains of Plasmodium vivax in the Aotus trivirgatus monkey were shown to be infectious to Anopheles freeborni and A. maculatus mosquitoes. The A. freeborni were more readily infected than were the A. maculatus with each of the strains. A comparison of the infection ratios indicated that each strain of malaria was different with regard to its mosquito infectivity. Transmission of the infection to 17 men was obtained by the bites of infected A. freeborni mosquitoes after extrinsic incubation periods of 13 to 18 days. The prepatent periods in the men ranged from 9 to 16 days with a mean of 13.8 days. The Aotus trivirgatus monkey has been shown to be potentially useful as an experimental animal in studies on the human malarias. Young et al. (1966) first reported infection of this monkey with a strain of Plasmodium vivax from Panama. Subsequent adaptation of P. falciparum and P. malariae parasites (Geiman and Meagher, 1967; Geiman and Siddiqui, 1969) has increased interest in the study of different isolates of the malaria parasites in the Aotus monkey. One area which has concerned us has been the infection of mosquitoes with malaria by feeding on these animals for subsequent transmission studies to man. Previously, we have reported the results of transmission studies using P. falciparum and P. malariae from the Aotus monkey (Contacos and Collins, 1968, 1969; Collins et al., 1968). Reported here are the results of studies with different isolates of P. vivax from Central America. MATERIALS AND METHODS The 4 strains of P. vivax used in the studies were obtained from naturally acquired infections from El Salvador (Sal-I, Sal-II), Panama (Panama), and Nicaragua (NICA), and provided to us by the staff of the Central America Malaria Research Station, NCDC, San Salvador, El Salvador. Once the strains were established in A. trivirgatus monkeys, they were maintained by serial passage of infected blood. Aotus trivirgatus monkeys, obtained commercially, had their origin in Colombia, South America. Prior serologic and microscopic examination indicated that they were free of natural malarial infection. Received for publication 21 October 1971. The Anopheles freeborni mosquitoes were the F-l strain originally isolated from Marysville, California, and maintained in the laboratory since then (Hardman, 1947). The A. maculatus mosquitoes were obtained from the Institute of Medical Research, Kuala Lumpur, Malaysia (Ow Yang et al., 1963). The techniques used for the feedings on the monkeys and on the human volunteers are those previously reported (Collins et al., 1968).

Plasmodium inui, a quartan-type malaria parasite of Old World monkeys transmissible to man.

A quartan-type parasite of Old World monkeys has been found infective to man by the bites of infected mosquitoes (Anopheles stephensi and/or A. maculatus) in two volunteers and by the inoculation of parasitized blood in five volunteers. The prepatent period in the sporozoite-induced infections was 31 and 56 days with parasitemia for 21 and 24 days, respectively. The blood-induced infections were patent for 10 to 26 days. A quartan fever pattern was well marked in two volunteers with maximum fever of 103.2 F. The incidence of chills was low. Parasitized blood from four of the volunteers when injected into clean rhesus monkeys produced typical infections. In 1960 Eyles et al. reported the first natural transmission of a simian malaria to man which occurred accidentally with the B strain of Plasmodium cynomolgi, a tertian-type parasite. This was followed shortly by the transmission of other strains of the same species by Coatney et al. (1961), Schmidt et al. (1961), and later by Bennett and Warren (1965). Contacos et al. (1963) transmitted P. brasilianum, a quartan-type parasite of New World monkeys, to man by the bites of infected mosquitoes. Chin et al. (1965) reported the first recognized transmission of P. knowlesi to man in nature. In addition, these authors were able to infect rhesus monkeys by the inoculation of sporozoites obtained from mosquitoes which became infected after feeding

Studies on human malaria in Aotus monkeys. I. Sporozoite transmission of Plasmodium vivax from El Salvador.

Monkey-to-monkey transmission of a strain of Plasmodium vivax from El Salvador was obtained on 12 occasions via the bites or intrahepatic and/or intravenous inoculation of infected salivary glands from Anopheles freeborni, A. albimanus, A. maculatus, and A. balabacensis balabacensis mosquitoes. The prepatent periods ranged from 14 to 48 days. The mean prepatent period was 34 days in intact monkeys and 29 days in splenectomized animals. The transmission rate was approximately the same in intact (57%) and splenectomized (53%) monkeys. Since Porter and Young (1966) reported the successful infection of Aotus trivirgatus monkeys with Plasmodium vivax from Panama, there has been a great deal of interest in the use of these animals as biological models for many different malarial studies. Young et al. (1966) and Porter and Young (1966) reported the successful transmission of P. vivax from the owl monkey to man via the bites of Anopheles albimanus mosquitoes. Subsequently, Baerg et al. (1969) reported transmission of P. vivax from A. trivirgatus and Ateles fusiceps to A. fusiceps, A. trivirgatus, and Saguinus geoffroyi via the bites of A. albimanus mosquitoes. Ward et al. (1969) showed that the Chesson strain of P. vivax could be transmitted with relative ease to A. trivirgatus monkeys via the bites of A. balabacensis balabacensis, A. stephensi, and A. quadrimaculatus mosquitoes. Since 1966, we have studied a number of different isolates of the human malarias in Aotus monkeys. This paper, the first of a series, reports the results of attempts to transmit a strain of P. vivax from El Salvador from monkey to monkey by sporozoite inoculation. MATERIALS AND METHODS The Sal II strain of P. vivax was isolated from a natural infection in the area of Las Guarumas, in the state of La Paz, El Salvador. Blood was inoculated into an A. trivirgatus monkey (AO-219) which was then provided to us by the staff of the Central America Malaria Research Station, CDC, San Salvador. The strain has been maintained by Received for publication 9 March 1973. * Center for Disease Control, Malaria Program, 1600 Clifton Road, Atlanta, Georgia 30333. serial blood or sporozoite passage in A. trivirgatus monkeys. The A. trivirgatus monkeys were obtained commercially, their origin being Colombia, South America. Prior parasitologic and serologic examinations indicated that they were free of natural malarial infection. Some of the animals were splenectomized prior to infection according to the technique of Sodeman et al. (1970). The A. freeborni mosquitoes were originally from Marysville, California, and have been maintained in the laboratory since 1944. The A. maculatts were obtained from the Institute for Medical Research, Kuala Lumpur, Malaysia, in 1964 and have been maintained in our laboratory since then. The A. balabacensis balabacensis colony was originally from Thailand and was obtained from the Walter Reed Army Institute of Research. The A. albimanus were the Apastepeque strain from El Salvador and obtained from CAMRS, San Salvador, in 1971. For the transmission attempts, sporozoites were introduced either by the bites of infected mosquitoes or by the intrahepatic and/or intravenous inoculation of sporozoites from dissected salivary glands. The technique for the intrahepatic inoculation has been reported previously (Held et al., 1967; Sodeman et al., 1969). For the transmission attempts by mosquito bite, mosquitoes were individually caged and allowed to feed on the restrained monkey. After biting, the mosquitoes were dissected and the salivary glands examined for the presence of sporozoites. The gland infections were graded 1+ (1 to 10 sporozoites), 2+ (11 to 100 porozoites), 3+ (101 to 1,000 sporozoites), or 4+ (greater than 1,000 sporozoites). Daily blood smears were made beginning 7 days after sporozoite inoculation to determine the prepatent period. Blood smears were stained with Giemsa.

Drug-resistant falciparum malaria from Cambodia and Malaya

Abstract 1. 1) Five strains of P. falciparum suspected of being chloroquine resistant were established in eight prisoner volunteers. Two of these strains originated from Cambodia and are referred to as Cambodian I and II. Three are from natural infections in Malaya and are referred to as Malayan I, II and III. 2. 2) These strains were studied for their response to one to five anti-malarial drugs; namely, chloroquine, proguanil, mepacrine, pyrimethamine and quinine. 3. 3) The Cambodian I strain was susceptible to chloroquine when the 1.5 gramme regimen of the base was given. 4. 4) The Cambodian II and the Malayan II strains were resistant to chloroquine, proguanil, mepacrine and pyrimethamine but susceptible to quinine. 5. 5) The Malayan I strain was resistant to chloroquine and to proguanil but susceptible to pyrimethamine and to quinine. 6. 6) The Malayan III strain was resistant to chloroquine, proguanil and pyrimethamine but susceptible to mepacrine and to quinine. 7. 7) Quinine was the only anti-malarial to which all strains were susceptible. A comparison between these strains and the chloroquine resistant strains from Colombia and Thailand is presented.

Clinical and physiological responses in sporozoite-induced B strain Plasmodium cynomolgi and Plasmodium vivax infections in normal volunteers

Abstract 1. 1) Five inmate volunteers were experimentally infected by sporozoite inoculation with the B strain of P. cynomolgi . The course of their infection was contrasted with that of two volunteers infected with the Venezuelan strain of P. vivax . 2. 2) The group of patients infected with the B strain of P. cynomolgi demonstrated a prepatent period of 9–15 days, an incubation period of 10–17 days, with the duration of their febrile illness lasting 8–12 days. The maximum parasite density in this group was 1100 parasites/c.mm. 3. 3) The patients infected with P. vivax demonstrated a prepatent period of 14 days, with an incubation period of 15 days. The duration of their febrile illness was 19 days, with the maximum parasite density controlled to 16,770/c.mm. 4. 4) Both groups of patients exhibited tertian fever patterns, but with the fever rising higher and lasting longer in the P. vivax infected patients. Headache, abdominal pain, and hepatosplenomegaly were prominent in the B strain P. cynomolgi infections. Generalized malaise, anorexia, weight loss, and shaking chills were pronounced in the P. vivax infected volunteers. 5. 5) Significant laboratory findings in both groups of patients were anaemia, leucopenia, thrombocytopenia, an elevated erythrocyte sedimentation rate, and shortened survival time of labelled red blood cells. The group infected with P. vivax demonstrated a moderately severe anaemia with a depressed reticulocyte response in comparison to the group infected with the B strain of P. cynomolgi . 6. 6) Other positive findings were hypoalbuminaemia and hyperglobulinaemia in both infections and a fall in the excretion of 17-hydroxycorticosteroid excretion in three of the five volunteers infected with the B strain of P. cynomolgi .

Studies on human malaria in Aotus monkeys. III. Exoerythrocytic stages of the Salvador II strain of Plasmodium vivax.

Exoerythrocytic stages of the Salvador II strain of Plasmodium vivax were demonstrated in sections of liver tissue from Aotus trivirgatus monkeys following the intrahepatic inoculation of sporozoites from Anopheles freeborni and A. maculatus mosquitoes. Six-, 7and 8-day forms were seen. In general, they appeared to be morphologically similar although smaller than those reported from

Fine Structure of the Exoerythrocytic Stage of Plasmodium cynomolgi

The light microscopy of primate exoerythrocytic stages (liver stage) of malaria is well described. This report demonstrates the ultrastructure of the 7-day exoerythrocytic stage of Plasmodium cynomolgi in the liver of a rhesus monkey.

Studies on human malaria in Aotus monkeys. V. Blood-induced infections of Plasmodium vivax.

Blood-induced infections of Plasmodiulw vivax in splenectomized Aotus trivirgatus monkeys produce peaks in the parasitemia an average of once every 42 days. Associated with these peaks are infections in Anopheles freeborni mlosquitoes which are highest in the 4 days before and including the peak in the parasitemia. Higher levels of mosquito infection and higher numbers of oocysts per gut were obtained in mosquitoes fed on monkeys splenectomized after a short period of parasitemia than in those splenectomized prior to inoculation. Intact monkeys infected mosquitoes at a very low level only. The Aotus trivirgatus monkey has been shown to be a useful host for studies of Plasmodiumn vivax (Young et al., 1966; Porter and Young, 1966). Of necessity, most of our early studies have been with animals infected by the inoculation of parasitized blood; these have been studied for the course of the parasitemia, both in intact and splenectomized animals, and for the infection of mosquitoes. Reported here are the results of some of our observations on blood-induced infections in these animals. MATERIALS AND METHODS A total of 70 monkeys infected with 14 different strains of P. vivax were examined. Twenty monkeys were infected with strains from Southeast Asia, 10 with Chesson strain (from New Guinea), and 40 with strains from Central and South America. The results were pooled since there appeared to be as much variation in parasitemia and infectivity between different monkeys as was seen between the different strains. All monkeys were the A. trivirgatus griseimnemibra subspecies originally from Colombia. Prior parasitologic and serologic examination indicated that they were free of natural malarial infection. All monkeys were infected by the intravenous inoculation of either fresh or frozen heparinized parasitized blood. During the early course of the infection, thick and thin blood films were made daily and stained with Giemsa for microscopic examination. Later, especially during periods of latency, smears were made at less frequent intervals. Whenever mosquitoes were fed, gametocyte counts were made on a daily basis. Anopheles freeborni mosquitoes were fed on the restrained animals between 0800 and 1200 hr. Incubation was at 25 C and 10% Karo syrup soluReceived for publication 4 December 1973. * Center for Disease Control, Tropical Disease Program, 1600 Clifton Road, Atlanta, Georgia 30333. tion was provided daily. Dissections of the guts were made between the 7th and 10th days after feeding and the total number of oocysts present on each gut was recorded. Splenectomy of monkeys was made according to the technique of Sodeman et al. (1970). RESULTS The monkeys can be divided into three groups: (1) intact animals, (2) monkeys splenectomized prior to infection, and (3) monkeys splenectomized after the appearance of parasites. Intact monkeys Twenty-eight monkeys were inoculated. The peak in the parasitemia was obtained between the 6th and 29th days after infection (mean 15.4 days). The peak parasitemia ranged from 20 to 720,000 per mm3 with a mean of 41,400 per mm3. Since the parasitemias thereafter usually fell to very low levels, most of the monkeys were splenectomized soon after the primary peak. Only one animal had a second peak in the parasitemia and this occurred on day 42. Presplenectomized monkeys A total of 42 monkeys were inoculated from 1 to 332 days after splenectomy (mean 62 days). The recrudescences in the parasitemia which could be identified by distinct peaks in the parasitemia are presented in Table I. Three of the animals had seven recrudescences, the longest interval between inoculation and final peak parasitemia being 319 days. The mean peak parasitemia was highest during the primary attack and continued to drop with each succeeding recrudescence. It is important to note that the reason some animals did not