The American Journal of Tropical Medicine and Hygiene - Volume 15, Issue 5, September 1966

A 2-year study was conducted in Haiti to determine the effect of the residual fumigant technique on Anopheles albimanus and on malaria transmission.

From 4,618 to 5,760 houses in the Commune of Arcahaie were treated with dichlorvos-wax dispensers at 3- to 4-month intervals and at a rate of one unit per 165 to 300 cubic feet of space. Twelve-hour exposure tests with caged A. albimanus females showed that the treatment gave average kills of 90 percent for approximately 12 weeks. Although malaria incidence declined, transmission was not interrupted, a result considered to reflect the tendency of A. albimanus to bits out-of-doors.

Behavior studies indicated that 75 to 80 percent of the human biting occurred outside of the house. Half of this feeding was between 5:30 and 7:30 p.m. during a period when the human population normally was outside of the house. These habits of A. albimanus also preclude the successful use of other types of residual house treatments in inter-rupting transmission. This study was a cooperative endeavor of the Agency for International Development, Pan American Health Organization, Communicable Disease Center, and the Government of Haiti.

Blood film samples were taken of 30 percent of the population in the Commune of Arcahaie, Haiti, to determine the effect on malaria transmission produced by the periodic treatment of homes with the dichlorvos residual fumigant. Prior to insecticidal treatment, infants 0–12 months showed a positivity index of 6.5 percent in 2,489 smears. At 1 and at 2 years after treatment, samples in the same age group had positivity indices of 7.0 and 2.2 percent, respectively. Plasmodium falciparum was the principal parasite but infections of P. vivax and P. malariae also occurred. Despite reduction in infection levels in infants and in children over 1 year of age interruption of malaria transmission was not apparent.

Complaints of headache, skin rash, and diarrhea among workers who unpacked and distributed dichlorvos dispensers for malaria control led to a study of men who did the same kind of work in the next cycle of application. Mild to moderate depression of plasma cholinesterase in two groups of workers corresponded with their degree of exposure. Red cell cholinesterase was not affected. There was no recurrence of the complaints that led to the study, and no signs or symptoms of intoxication by an organic phosphorus compound were discovered.

The characteristics of the immune response observed during the primary attack of Plasmodium vivax malaria were similar in sporozoite-induced and blood-induced cases. In both types of infections antibody production closely followed the appearance of parasites in the peripheral circulation, antibody levels abruptly rose to a maximum, stayed at high levels for about a month, and, after elimination of the parasites, gradually declined but persisted at low levels for extended periods of time. The close correlation between the appearance of the asexual forms of the malaria parasite and the first detection of antibody suggested that the asexual stages provided the antigenic stimulus. Evidence is presented which suggests that the gametocytes probably do not initiate the immune response. Results of studies in several blood-induced cases given many dead organisms suggested that killed parasites may be capable of provoking a mild antibody response. A pronounced booster effect, associated with the secondary antibody response, was observed in several volunteers who relapsed. Despite intermittent drug therapy given relatively early in the infection, all of the vivax cases under study exhibited an appreciable antibody response.

The course of sporozoite-induced Plasmodium berghei malaria was studied in albino rats, albino mice, golden hamsters, and tree rats (Thamnomys surdaster), a natural host of this parasite. Animals were inoculated with sporozoites obtained by trituration and differential centrifugation of laboratory-bred Anopheles stephensi which 14 days previously had fed on hamsters infected with the NK 1965 strain of P. berghei.

After a uniform dose (25,000 sporozoites injected intraperitoneally) the course of infection was characteristic for each rodent species and with the exception of the mice showed little variation from experiment to experiment.

The injection of smaller inocula (10,000 and 1,000 sporozoites) not only reduced the percentage of infected animals within each species but also delayed the course of the resulting infections. This was generally refected in their longer prepatent periods, delayed onset of parasitemia, and longer survival. Young rats were shown to be the most susceptible animal to sporozoite-induced infection.

The first known transmissions of Plasmodium ovale by Anopheles freeborni and A. maculatus are reported. The successful transmission of P. ovale by A. freeborni is of interest because this is the the first reported vector which can transmit all 4 species of human malaria.

An “indirect” fluorescent antibody test for antibodies in cases of amebiasis is described. Antigen consists of washed, intact, formalin-fixed amebae from cultures growing with mixed bacteria. Counter-staining with Evans Blue greatly improves ability to determine end-points in titrations.

Antigen from the Huff strain of Entamoeba histolytica, capable of growing at reduced as well as at body temperature, showed far fewer positive reactions than antigen prepared from a virulent, “classic” strain of this species. With the latter antigen, 73.1% of cases with confirmed intestinal amebiasis, and 91.3% of cases with confirmed or unconfirmed extra-intestinal amebiasis were found positive at 1:16 or higher. However, 5 out of 16 individuals without amebic infection were also found positive at low titer. A possible explanation for the false positives and a means for eliminating it are suggested. It is concluded that the test as here presented is technically well-fitted for the experienced laboratory to use, with caution, until more information is accumulated to explain the few anomalous results.

In a review of iron-hematoxylin preparations from a survey for amebic infection in U. S. State Department employees and patients at the Clinical Center, National Institutes of Health, four cases of infection with cysts of Entamoeba polecki were found. It was estimated that the incidence of occurrence of infection in these populations was about 1:3000. Only 1 of the 4 cases detected occurred in material previously identified as infection with E. histolytica, while the others were found in smaller numbers of cases identified as probable infection with E. histolytica or Entamoeba species, unclassified.

Fifty-four primates were exposed to single graded doses of 50 to 2000 cercariae of Schistosoma mansoni (Puerto Rican strain). They belonged to the following ten species: Macaca mulatta (rhesus monkey), Macaca cynomolgus (irus monkey), Macaca speciosa (stump tail monkey), Cebus apella (capuchin monkey), Ateles geoffroyi (spider monkey), Papio anubis (baboon), Pan satyrus (chimpanzee), Callithrix aurita (marmoset), Saimiri sciureus (squirrel monkey), and Tupaia sp. (tree shrew).

While all of these species could be experimentally infected with S. mansoni, their susceptibility and course of infection varied considerably. Variations in the intensity and type of tissue responses were also observed at various dosage levels. In general, three major reaction patterns could be distinguished: In the rhesus, irus and stump tail monkeys, there was a high percentage recovery of worms, good worm development and numerous infective eggs widely distributed throughout the colon, small intestine and liver. After a few months following infection, in these animals there was a tendency toward gradual self-cure. In the baboon and chimpanzee there was a lower worm recovery but egg excretion was maintained over relatively long periods. Most of the eggs were in the colon. In the marmoset, squirrel monkey and tree shrew, the infection tended to be aborted from its onset. The worm and egg recoveries were low, the miracidial infectivity for snails was also low or absent and the pathological features were irregular and atypical. There is not yet sufficient available evidence to determine the exact position of the capuchin and spider monkeys.

None of the primates included in these studies developed pipe-stem fibrosis of the liver in any way similar to that of man except for the chimpanzee which showed moderate portal fibrosis and development of portocaval collateral anastomoses. The liver architecture was generally preserved even among the monkeys of the third group which had large and fibrotic pseudotubercles.

No obvious correlation could be observed between susceptibility, phylogenetic position and the habitat of the primates studied.

A skin test evaluation for schistosomiasis was made on a citrus estate located near Salisbury, Southern Rhodesia. One hundred fifty-five Bantu children, 6–13 years of age (100 males and 55 females), were tested. In this group, 142 (92%) were found to be infected with either Schistosoma mansoni or S. haematobium on examination of one urine and one stool specimen. The group was tested with antigens prepared from adult worms of S. mansoni administered by needle and syringe. In addition, the same antigen was administered with a foot-powered hypodermic jet injection apparatus equipped with an intradermal head. Antigens prepared from adult worms of S. japonicum and larvae of Trichinella spiralis were administered and tested by needle and syringe in the skin. Control antigen consisted of buffered saline with merthiolate.

The average wheal area obtained in the group with the control antigen was 0.6 cm2; with the S. mansoni antigen (needle and syringe) 3.0 cm2; with the S. mansoni antigen (jet injector) 2.5 cm2; with the S. japonicum antigen 2.9 cm2; and with the T. spiralis antigen 0.8 cm2.

In the group, 25 were infected by S. haematobium, 50 by S. mansoni and 67 by both parasites. There was a significant difference in skin reactivity demonstrated by individuals infected with one or both parasites. There was no significant difference in the average size of the wheal area between S. mansoni and S. japonicum antigens. The S. mansoni antigen was, however, more sensitive than the S. japonicum antigen. Five of 142 infected individuals were false-negative with the S. mansoni antigen, whereas 10 of 142 were false-negative with the S. japonicum antigen.

The excretion of S. haematobium eggs in the urine over a one-hour period was measured. There was no correlation between the size of the wheal in the skin test and the hourly excretion of eggs.

Three individuals out of 88 tested with the T. spiralis antigen were positive with wheal areas of 1.4, 1.9 and 3.0 cm2. The serum of the individual with the 3.0 cm2 wheal area was tested for antibodies against T. spiralis and was found to be negative.

Eggs of S. mansoni were found in the urine of eight individuals, eggs of S. haematobium in the stools of two individuals and eggs of S. matheei in the urine of one individual. Of the 13 individuals negative by one urine or stool specimen, seven were positive by the skin test.

A random sample of 19 Puerto Rican patients infected with Schistosoma mansoni were treated with a 40- to 80-mg dose of Astiban®/kg of body weight over periods ranging from 11 to 16 days. In spite of the high cure rate of 94%, the frequency and severity of the side effects do not justify the use of this massive dose in schistosomiasis chemotherapy.

Fifteen additional species of animals collected from central and northeastern Thailand were found naturally infected with the third-stage (infective) larvae of Gnathostoma spinigerum, bringing the total to 36 known species of natural second intermediate hosts, of which fresh-water fishes (Ophicephalus and Clarias), the domestic duck and the domestic chicken probably constitute the more important sources of infection in man. Experimental infections suggest 20 species of animals as potential second intermediate hosts even though not all have been found naturally infected. Infective larvae were usually first located in the liver, later in the muscles. The hamster, Norway rat, white rat, white mouse, guinea pig, domestic chicken and domestic duck are susceptible to the infection and can be used effectively as laboratory hosts.

“Medusa-head” agglutination phenomenon was for the first time observed, reproduced and recorded in vitro in citrated blood of a case of Loa loa which exhibited a low microfilaremia. These microfilariae were oriented tail in and head out to the configuration. Within the clump the posterior end of each microfilaria was bent sharply toward the anterior and against the body of the parasite.

No definite conclusions could be drawn from these observations but the posterior bending and attachment at this site suggest either a specific physical-chemical attraction or a possible antigen-antibody reaction of this portion of the microfilaria to the attached fibrin mat, to the exclusion of other areas of the organism.

Results of one year's study of leptospirosis in Nicaraguan wild and domestic animals is presented. Leptospirae of the canicola group were isolated from Urocyon cinereoargenteus, Spilogale putorius and Conepatus semistriatus; of the pyrogenes group from Oryzomys caliginosus; serotypes rama and abramis and leptospirae of hebdomadis and autumnalis groups from Philander opossum. These investigations support the view that wild animals are unimportant in the episootiology of clinical bovine leptospirosis.

An investigation of the bacterial and parasitic agents associated with diarrheal illness in 655 children under two years of age was conducted in Karachi, West Pakistan. Four hundred sixty-eight children of similar age but without diarrhea were studied as controls. Specimens were obtained from both hospitalized patients and out-patients.

Enteropathogenic bacteria were isolated from 19.6% of all diarrhea cases and 4.7% of all controls. Shigella, the commonest bacterial pathogen, was recovered from 12.4% of children ill with gastroenteritis and 1.3% of controls and showed an increasing incidence with age up to two years. Enteropathogenic Escherichia coli was found 2.5 times as often in diarrhea cases as in controls, and most frequently in the age groups from birth to 6 months and from 13 to 18 months. E. coli serotype 0126:K71 (B16) was the most common in the hospital cases and 0127:K63 (B8) in the clinic cases. Salmonella was found in less than 0.5% of all children.

Nutrition appeared to be an important factor in the children with diarrhea. Approximately 50% of hospital patients showed some degree of marasmus or kwashiorkor. Onset of diarrheal episodes appeared to be associated with weaning.

Parasites were found in equal incidence in diarrhea cases and controls with the exception of Giardia lamblia, which was found 1.6 times as often in children with gastrointestinal symptoms.

Although hemorrhagic fevers (HF) have been extensively treated in scientific literature of the U.S.S.R., these diseases were of limited concern to Soviet health authorities until 1965 when a regulation to make them reportable was introduced. Our discussions with medical and other scientific authorities suggested that HF incidence is higher and exposure risk is much greater than we had anticipated on the basis of official reports; furthermore, it seemed that the number and variety of recognized natural foci are on the increase.

Hemorrhagic fever with renal syndrome (HFRS) is by far the most important epidemic HF in the Soviet Union today. Omsk HF (OHF) has been of little recent importance, except among workers in the muskrat for industry. Crimean type HF (CTHF) has been absent from the Crimea for many years; a very similar epidemic disease presumably caused by the same virus now occurs in the Volga and Don delta regions. Central Asian HF (CAHF) was said to be practically non-existent at present, possibly because of environmental modifications by extensive irrigation systems.

Appraisal of the HF classification widely accepted in the U.S.S.R. exposed many lacunae in clinical, virological, epidemiological, ecological, and zoological knowledge of Soviet HF entities (Table 2). This classification should therefore be considered a tenuous synthesis of certain demonstrated facts and speculations based on field observation. An especially important problem is whether HFRS, with its high morbidity and mortality in the Far East, is the same etiologic entity as HFRS in the European U.S.S.R., where it is a relatively mild disease (this is of particular interest because of the assumed relationship of Korean HF and HFRS). The question will not be definitely answered until etiologic agents have been isolated and characterized.

At this time OHF is the only characterized HF syndrome for which an undisputed etiological agent is available. The status of etiologic agents for other HF entities continues to be unsettled. In 1964 the GON virus strain was isolated from the blood of a patient suffering from what was clinically diagnosed as CTHF; forming plaques in its 19th passage in cercopithecus monkey kidney cell cultures, this strain is currently the most promising agent for CTHF, although its role has not been yet definitively established. Repeated and complicated virological attempts to isolate the etiological agent of CAHF have proved fruitless. Isolation of an HFRS agent from human volunteers was reported in the early literature, but extensive attempts to recover viruses from specimens collected during recent outbreaks have failed.

The recognized inadequacy of investigations on HF pathogenesis and pathophysiology elsewhere in the world appears to be also true for the U.S.S.R.; this includes even OHF, the etiology of which was established 20 years ago. Zoological and ecological studies on the muskrat and vole (Omsk) and on rooks and domestic mammals (Volga and Don deltas) are examples of field investigations that need to be multiplied and expanded to elucidate the HF problems. Owing to their biological and microbiological implications, ticks, especially of the genus Hyalomma, require more extensive field surveys and collections to allow further bionomic and systematic assessment.

The Delegation was exceptionally well received in the U.S.S.R. and, in retrospect, the mission was remarkably successful. Unfortunately, due to conditions beyond our control, it was not possible to assess fully the data on HFRS and OHF. What was learned emphasised the desirability of a more deliberate examination of these problems in the recognized foci and regional laboratories at Ufa, Omsk, Khabarovsk, and Vladivostock, even though such undertaking would require another month. Best use was made, however, of available opportunities to examine the natural foci and investigations in progress on CTHF in the Rostov and Astrakhan regions of the Russian republic and on CAHF in the Kazakh and Uzbek republics of Central Asia.

A lot of formalin-inactivated Japanese B encephalitis vaccine was prepared from an attenuated strain of OCT-541 virus, grown in hamster kidney cell, in medium 199 and 2% human albumin at pH 7.9. The pre-inactivation infectivity and hemagglutination titers were 108.3/0.1 ml and 1:256 respectively after Millipore filtrations. The virus suspension was inactivated with 1:4000 formalin at 37°C for three times the period estimated for destruction of measurable infectivity. Sterility and safety tests showed that the final vaccine was free from bacteria and other detectable adventitious agents. The potency of the vaccine was established by the mouse test (minimal immunogenic dose of 0.0089 ml) and by the development of high anti-body titer in immunized guinea pigs and monkeys (neutralization index of >4.8 logs). One vaccinated monkey observed for 9 months maintained a high antibody level for this period.

Data regarding the vaccine stability were presented for other lots with longer storage, which suggested that freezing did not lower the potency and the one preparation frozen at about -70°C and observed for about 14 months maintained its potency for that period.

Four of 46 weak and nonflying Mexican free-tail bats (Tadarida brasiliensis mexicana) captured in Frio Cave, Uvalde County, Texas, were found to be infected with bat salivary gland virus. One of these four bats survived and was maintained in a nonhibernating state in the laboratory, and bat salivary gland virus was recovered from the saliva on 10 occasions over a period of 309 days after the bat was captured.

Nepuyo virus, represented by one strain from Trinidad and five from Brazil, reacts by hemagglutination-inhibition (HI), neutralization and complement-fixation (CF) testing with previously described group C types but is distinct from them. By HI and neutralization testing Nepuyo is most closely related to Murutucu virus, and by CF testing to Apeu and Marituba viruses.

The mosquito Aedes aegypti was infected with Semliki Forest virus, both by feeding on viremic mice and by the injection of virus into the hemocoele.

Transmission of virus to suckling mice was demonstrable within a week after infection, but the transmission rate then declined to reach zero after three weeks, in spite of the fact that the thorax still contained large amounts of virus.

The salivary glands of infected moequitoes showed cytological changes which were maximal three weeks after infection. At this time mosquitoes had some difficulty in obtaining a blood meal. The salivary gland changes included a gross decrease in the secretory regions of glandular cells, and an inability of these cells to exclude trypan blue.

The salivary gland changes occurred in mosquitoes infected by feeding or by injection with high or with low doses of virus.

No shortening of the life span of infected mosquitoes could be demonstrated.

Attempts to study the salivary glands of infected mosquitoes by fluorescent antibody staining failed.