The American Journal of Tropical Medicine and Hygiene - Volume 17, Issue 2, March 1968

Members and guests of The American Society of Tropical Medicine and Hygiene:

Each of the 31 years since 1936 the Society has taken cognizance of its debt to and esteem for Col. Charles Franklin Craig. This recognition has followed the form of a named lecture presented by one whose selection not only reminds the Society of Col. Craig's contribution to tropical medicine, but also serves as formal recognition of the accomplishments of the Lecturer himself. The 32nd-year's participant in this two-way reaction is Dr. G. Robert Coatney.

Dr. Coatney is so well known to this audience that introducing him presents serious problems, analogous in some respects to presenting President Lyndon B. Johnson to the United States Senate. Few members have played more different roles in the affairs of the Society than this year's Craig Lecturer—and I might add, none with more enthusiasm and distinction.

The Committee, in giving me the opportunity to deliver this Charles Franklin Craig Lecture, has paid me a signal honor. The high standard set by the distinguished speakers who have preceeded me in this role should have deterred me, but casting caution aside—I accepted. I hope the Committee will not lose face as a result of my decision and that the Society will not only survive but also continue to flourish.

There is little doubt that simian plasmodia were seen around the turn of the century, although the first detailed description did not appear until 1907. As descriptions of other species appeared, it was realized that many of them were morphologically similar to the malaria parasites of man. It is not surprising, therefore, that attempts would be made to set up infections with these parasites in man. Blacklock and Adler in 1922 carried out the first trial using Plasmodium reichenowi. It failed.

The fine structure of the exoerythrocytic stages of Plasmodium gallinaceum was studied in chick-embryo liver and was compared with that of the exoerythrocytic stages of avian malaria parasites in a tissue-culture system. The structure of the parasite in this present study seems to be essentially similar to that grown in vitro. These observations appear to confirm the usefulness of the tissue-culture system for studies of the development of the exoerythrocytic stages of avian malaria parasites, at least from the standpoint of fine structure.

The exoerythrocytic stages of P. gallinaceum in chick-embryo liver are found within the endothelial cells of the sinusoids, an observation coinciding with the findings obtained by light microscopy. As the parasite grows, the endothelial cell is displaced by the large parasite and protrudes into the sinusoidal space. At the end of merozoite formation, the endothelial cell forms a thin shell around a large cyst containing several merozoites. The possibility that the membrane surrounding the parasite in the endothelial cell may be derived from the pinocytotic vesicles or from the endoplasmic reticulum of the host cell was discussed. Occasionally degenerating parasites were observed within a digestive vacuole of the Kupffer's cell. The hepatic cells of chick-embryo liver in general seem not to be affected by the infection with the exoerythrocytic stages of P. gallinaceum, except by the increased number of lipid droplets in the cytoplasm.

Normal and splenectomized rhesus monkeys were transfused with 51chromium-labeled erythrocytes from monkeys severely infected with Plasmodium knowlesi. Radiochromium disappeared from the blood of both normal and splenectomized recipient animals at a rapid and similar rate. Parasites disappeared from the blood of normal but not splenectomized monkeys faster than did the radiochromium. This suggested that the spleen removed malaria parasites from transfused cells without destroying the erythrocytes that contained them.

In three experiments it was shown that mice that had recovered from P. chabaudi infection were resistant to challenging B. rodhaini infections. Mice that were challenged 1 week after recovery appeared to be more resistant than those challenged after 5 weeks. On the other hand, mice recovered from P. chabaudi appeared to be as susceptible as control mice when they were challenged with P. berghei. There was, however, in each of three experiments a 2- to 3-day delay in the development of P. berghei parasitemia in the mice recovered from infection with P. chabaudi.

In two experiments it was evident that rats that had recovered from P. berghei infection were resistant to challenge with B. rodhaini. Resistance of rats in the converse situation is less clear since the resistance of rats recovered from infection with B. rodhaini was obscured by extremely high P. berghei parasitemia in three of 10 recovered rats.

The results of these experiments suggest that part of the immunizing mechanisms in both malaria and babesiosis are nonspecific and that the same mechanism probably pertains in both infections. It is suggested that the serum antigens in acute infections, which have been shown to be both nonspecific and immunogenic in previous work, are primarily responsible for the observed results.

The fact that mice that had recovered from P. chabaudi infection resisted B. rodhaini, but did not resist with P. berghei does not indicate that P. berghei is a more virulent parasite. In our experience B. rodhaini is as lethal for mice as is P. berghei. We believe that the apparent lack of resistance of these mice to P. berghei is a manifestation other mechanisms, that is, the selection of “immunity-resistant” variants or mutants by the immunized mice.

An adult female D. tenuis containing microfilariae was found in the subcutaneous tissues of the abdominal region of a man from Florida. Structures resembling dead microfilariae were seen within nearby granulomata. The eosinophilia, antibody titers, and apparent response to diethylcarbamazine suggest occult filariasis similar to that seen in Southeast Asia.

The bacteriologic and pathological characteristics of four fatal cases of melioidosis are reported. Pseudomonas pseudomallei was cultured from the following specimens: 1) ante mortem—sputum, skin lesions, blood, synovial fluid, and bone marrow; 2) post mortem—lung, meninges, heart blood, spinal fluid, pleural cavity, and skin lesions. Eosin-methylene blue agar was found to be the medium of choice for primary isolation. All isolates of the organism were homogeneous in their biochemical and serologic characteristics as were their sensitivities to a variety of antibiotics and a sulfa drug.

Post-mortem examination in Case 4 showed gross and microscopic lesions in the lungs, skin, and kidneys. Microscopic lesions were also found in the liver and spleen. In Cases 1, 2, and 3 there were gross lesions in the lungs, skin, kidneys, liver, spleen, subcutaneous tissue, lymph nodes, and meninges. These were confirmed microscopically.

The repository sulfone 4,4′-diacetyldiaminodiphenylsulfone (DADDS) was studied for its efficacy in lepromatous leprosy. It was given in a dosage of 225 mg every 77 days to a group of 10 patients, who were matched with a group of 10 patients given oral 4,4′-diaminodiphenylsulfone (DDS, dapsone, diaphenylsulfone) in a dosage of about 100 mg per day.

Two chief criteria of therapeutic response were employed: reduction in numbers of M. leprae in nasal washings and reduction in the ratio of solidly staining M. leprae in skin smears (MI). Both criteria are thought to measure reduction in viability of the bacilli. By both measurements DADDS was as active as DDS. There were two deaths in the group receiving DADDS, but these were not thought to be connected with drug toxicity.

The blood level of “free” sulfones was usually less than 0.2 µg per ml in the patients receiving DADDS. The urinary output averaged 1.6 mg per 24 hours. It was estimated that the blood level remained several times higher than the minimum inhibitory concentration of DDS for M. leprae.

Adult cebus (Cebus sp.), patas (Cercopithecus patas), rhesus (Macaca mulatta), and vervet (Cercopithecus aethiops) monkeys were found susceptible to experimental infection with Leptospira icterohaemorrhagiae (as evidenced by febrile response, weight loss, antibody production, and recovery of leptospires from hemoculture of some of the animals), although they did not show frank illness. Vervets were similarly susceptible to infection with L. canicola, L. mini georgia, and L. pomona. Leptospiruria was uncommon.

Squirrel monkeys (Saimiri sciureus) given L. icterohaemorrhagiae had in contrast clinically acute leptospiral disease, sometimes fatal, with pronounced icterus and minimal hepatic lesions accompanied by markedly increased indirect bilirubin and normal transaminase levels. The findings suggest that hemolysis may be a significant factor in the early development of jaundice in these animals.

In some of the primates tested, the tendency of leptospiral agglutinins to disappear within a few months makes retrospective serologic studies for leptospirosis of doubtful validity. In the various species studied, except squirrel monkeys, L. icterohaemorrhagiae infections elicited the “paradoxical” serologic reaction, which further emphasized the difficulty of determining the infecting leptospiral serotype by serologic means.

Primary infection with L. icterohaemorrhagiae protected animals well against attempted homologous superinfection. Little cross-protection could be demonstrated by prior L. icterohaemorrhagiae infection against subsequent challenge by L. canicola or L. pomona.

The pathogenesis and spotted-fever-group complement-fixing (CF) antibody responses of black-tailed jack rabbits and cottontails following inoculation with Rickettsia rickettsii were investigated. Cottontails inoculated 1 month after being trapped did not have rickettsemia that persisted for as long as it did in cottontails 9 months after capture. Of nine jack rabbits tested, rickettsemia lasting for 6 days developed in two. There was a marked variation in the persistence of rickettsemia in the cottontails and jack rabbits tested. There did not appear to be any tendency for latent infection in cottontails, as rickettsiae did not persist for longer than 20 days in the tissues of the animals tested. CF antibody was readily found in jack rabbits, but the response in cottontails was variable, and some cottontails known to have been infected did not have CF antibodies injected with a soluble antigen.

It was concluded that the jack rabbit may not be as important in the cycle of R. rickettsii in nature as previously believed and that cottontails are of greater importance in the dissemination of R. rickettsii in nature.

Primary tissue cultures prepared from Aedes aegypti embryos were inoculated with Eastern equine encephalomyelitis, Semliki Forest, and West Nile viruses. The viruses multiplied in this in vitro system and were present in the cultures for periods as long as 60 days after infection.

The growth process for the three viruses proceeds differently: WN virus is the slowest growing, EEE is intermediate, and the SF virus grows fastest.

Cultures infected with WN virus did not show morphologic changes, whereas those inoculated with EEE or SF viruses occasionally displayed cellular alterations. There is no evidence that these cellular alterations can be attributed to virus infection.

The presence of EEE virus in cultures was also verified by the hemagglutination test, but no hemagglutinin was detected in cultures infected with WN and SF viruses.

Bush Bush Island and the surrounding Nariva Swamp, eastern Trinidad, are described in terms of their topography, flora, and fauna as an introduction to a series of papers reporting the results of 5 years of arbovirus studies in this area.

The field program of the Trinidad Regional Virus Laboratory in Bush Bush Forest included studies in which arboviruses (their prevalence and epidemiology) were the chief interest and also studies on the biology of the arthropods and vertebrates possibly associated with arbovirus cycles. The techniques used included the exposure of sentinel animals, trapping and bleeding of small mammals and reptiles, netting and bleeding of birds, collection of bloodsucking arthropods, and observations on the diel- and seasonal-activity patterns, food sources, and habitat preferences of the more abundant species of mosquitoes.

Bush Bush Forest entomologic investigations were concerned mainly with mosquitoes, the most conspicuous element of the bloodsucking arthropod fauna. More than 92 species were demonstrated, but only about two dozen were common. Mosquitoes were studied in relation to their physical environment, seasonal activity, diel activity, horizontal stratification in the forest, food preferences, and larval habitats. Large numbers were collected for virus studies.

Other groups investigated were phlebotomine flies (10 species), Culicoides flies (10 species), horseflies (18 species), Cuterebridae (one species), sucking lice (four species), fleas (one species), ticks (nine species), and various mites including 15 species of trombiculid.

An introduction has been given to the principal vertebrates of Bush Bush Forest, which include 59 species of mammal (32 of bat), 171 species of bird, 27 species of reptile, seven species of amphibian, and an undetermined number of fish. Special attention was paid to the population dynamics, longevity, and range of small forest rodents. During the study years the populations of small rodents declined almost to the point of disappearance.

Virus isolates totaling 593 and representing 19 different virus types were recovered from sentinel animals, small mammals, and arthropods during studies carried out in Bush Bush Forest, Trinidad, over a 5-year period. The laboratory techniques used are described, and details are given regarding 49 isolates belonging to 12 virus types: Una, St. Louis encephalitis, Ilhéus, Bushbush, TRVL 57896 (Moriche), Wyeomyia, Cache Valley, Cocal, Oropouche, Aruac, Nariva, and TRVL 39316-1-5 type (Pacui). The remaining 544 isolates were identified as Venezuelan equine encephalitis virus or members of serologic groups C and Guamá, and are discussed elsewhere.

Details are given on isolations of seven virus types from material collected in Bush Bush Forest during the study period: 262 of Venezuelan equine encephalitis (VEE), 71 of Caraparu-like, three of Oriboca, two of Restan, 63 of Bimiti, 56 of Catu, and 87 of Guamá. Data from experimental-infection studies are presented, as well as results of serologic studies with hemagglutinating antigens of VEE and Caraparu-like viruses and some 1,200 sera of Bush Bush rodents.

It was found that the natural transmission cycles of five of these virus types in Bush Bush are similar and include a period of multiplication and viremia in forest-floor rodents and a period of multiplication in mosquitoes among which Culex (Melanoconion) portesi, at least, is capable of subsequent transmission. Similar cycles are inferred for the remaining two virus types, Oriboca and Restan. In 1964 a severe reduction of the rodent population took place, Culex portesi continued abundant, and only Bimiti of the seven virus types was isolated. It is concluded that an alternate host population of sufficient size was not then available to these viruses in Bush Bush.

Very little evidence was found to implicate mosquito species other than C. portesi as essential vectors during epizootic or inter-epizootic periods.

Data obtained at the Bush Bush Forest field station in the Nariva Swamp, eastern Trinidad, indicated the continuous presence there of three Guamá-group agents—Bimiti, Catu, and Guamá—all apparently dependent on the same rodent host and vector populations. This suggested the possibility that the antigenic relation of these viruses might influence their epidemiology. The question was investigated by examining the effect of cross-immunity on circulating-virus levels in captive-reared Oryzomys laticeps and Zygodontomys brevicauda, two species of rodent that are natural hosts for these viruses.

Bimiti virus was circulated at lower levels in nonimmune Zygodontomys than were the other two viruses, and in cross-immunized Zygodontomys, Bimiti-virus levels were still lower and occasionally undetectable. Catu viremia reached high titers in both nonimmune Oryzomys and Zygodontomys, and even in cross-immunized rodents, titers often reached 2.4 to 3.3 log LD50. Guamá viremia in nonimmune Oryzomys and Zygodontomys was of the same order as that of Catu virus, but in cross-immunized rodents it was often completely suppressed.

These data suggest that when hosts are scarce Catu virus would be affected least and Guamá virus most. The experimental findings are compared with field observations.

Neutralization tests with Micrurus fulvius fulvius and Micrurus carinicauda dumerilii venoms indicate that antiserum specific for fulvius venom has greater cross-neutralizing potency than antiserum specific for carinicauda. However, the level of cross-protection of anti-carinicauda serum was high enough, and the carinicauda venom is available in sufficient quantity so that the venom could be used to produce antivenin for treatment of bites by fulvius.

Gel-diffusion reactions showed that carinicauda venom contained more precipitating antigens than fulvius venom; however, the venoms have several common antigens.