The American Journal of Tropical Medicine and Hygiene - Volume s1-22, Issue 5, September 1942

• 1.  Using a fluid medium consisting of buffered phosphate in sodium chloride solution, liver fluid, and rice starch as an encystment medium, a study has been made of the relation of hydrogen-ion concentration of the medium to the encystment of E. histolytica in culture.
• 2.  It has been found that the optimum total salt concentration in the medium for growth and encystation of E. histolytica is M/30 phosphate and 0.4 per cent sodium chloride.
• 3.  Utilizing the optimum phosphate and sodium chloride concentration, the optimum pH value of the medium for cyst production was determined. It was found that when the initial pH value of the medium was 6.8 to 7.0, and the pH values after 24, 36 and 48 hours of incubation were 6.7–6.9, 6.8–7.0 and 6.9–7.2 respectively, rich encystation was observed. The maintenance of a favorable pH range throughout the period of incubation is believed to depend not only on the initial pH value but also on the accompanying bacterial flora.
• 4.  It appears under the condition of these experiments that in order to have a rich encystment, even in a medium of favorable pH value, the rate of growth of the amoebae must also be greatly accelerated. When abundant growth is reached, the majority will excyst.
• 5.  Not all strains of amoebae give a rich cyst yield even when these two requirements are fulfilled. It is believed that all strains of amoebae have a tendency to encyst under favorable conditions, but much of this tendency may be lost on prolonged cultivation in a medium.
• 6.  The liver extract favors the growth of amoebae, but the amount of liver extract required to make up the medium, within the ratio of 1:60 to 1:160 to that of the phosphate-salt solution, affected the encystment only through its effect on the pH values.
• 7.  Amounts of encystment medium of 15–20 ml. seem to maintain a favorable pH value range better than smaller or larger amounts, and therefore induce better encystment.
• 8.  On the basis of this study, a medium is proposed for encystment of E. histolytica in culture. It is composed of 15–20 ml. of M/30 phosphate buffered at pH 7.6, ⅕–⅙ ml. of liver extract fluid, and 2–3 loopfuls of rice starch. With a strain known to encyst in culture and having a good accompanying bacterial flora, rich encystment is to be expected in this medium. To assure good results, the initial pH of the medium prepared should be tested and should not be lower than 6.8. If the medium is to be stored for some time before use, the phosphate-salt solution should be buffered at pH 7.8, as the pH value decreases on storage.

Rees (1937) published a description of a micromanipulator which he designed for performing the following operations: (1) Selecting individual protozoa from a mixed population of bacteria and protozoa, (2) transferring the protozoa to sterile fluid and thus eventually effecting complete separation of the fauna from the flora, and (3) seeding the isolated protozoa into tubes of sterile culture medium. The present paper is required because the earlier publication was inadequately illustrated and because several modifications have since been introduced.

ASSEMBLY OF THE MICROMANIPULATOR As stated by Rees (1937) the successful microisolation of protozoa from bacteria entails the following operations: (1) Accurate movement of the water-air meniscus in the micropipette, (2) movement of the pipette at will in the field of the compound microscope in any of the three dimensional planes, and (3) speedy removal of a contaminated pipette from its holder and its replacement with a sterile pipette.

Specimens of Mesocestoides, identified as M. variabilis, are reported from man for the first time. A detailed description of the human specimens is given. The human infection was probably derived from eating the improperly cooked flesh of a wild mammal or possibly a frog.

The development of economical and effective anti-hookworm procedures requires reasonably precise knowledge, not only of the incidence and intensity of infection, the personal habits of the people and the environmental conditions under which they live, but of their physical status as well. The program now in effect in Georgia emphasizes the hookworm-diseased family rather than the egg-positive individual as the unit of investigation and control. In practice, the selection of these hookworm-diseased families is made by local health or school personnel and is based, first of all, on physical evidence of anemia. As most of the secondary anemias in Georgia are due to or are augmented by dietary iron deficiencies, malaria or hookworm infection, the next step is to determine which of these factors or combination of them is responsible and to proceed with appropriate curative and preventive measures. In evaluating the rôle of hookworms, some quantitative approximation of the worm burden at which their anemia-producing effect becomes apparent is necessary for the guidance of local health agencies.

The name, helminthiasis of the intestinal wall, is proposed for pathological conditions resulting from invasion of the wall of the intestine by worms. This helminthic invasion leads to destruction of tissue, especially in the submucosa, hemorrhage, and concomitant inflammatory reactions. A description is given of 5 further cases of helminthiasis of the intestinal wall observed in autopsies in Batavia, Java, and caused by Ancylostoma duodenale. Necator americanus, by far the most common hookworm in Java, does not show this invasive power. Invasion of the intestinal wall usually takes place in the jejunum, but it may also occur in the ileum or even in the colon. There is no proof that the lesions thus produced endanger directly the life of the host.

In connection with other studies, a brief visit was made during 1941 to certain localities situated near the Rio Segovia in Northern Nicaragua. As no published information is available about the anophelines of that area, the following notes have been compiled to record what was found.

In Howard, Dyar, and Knab (1), the distribution of Anopheles crucians is given as the Southeastern United States from New York to Texas as well as Cuba and Jamaica. But King and Bradley (2) and Rozeboom (3) reported the presence of that species in Mexico; while Kumm and Ram (4) recorded it in British Honduras, and Clark (5) extended the known distribution to Spanish Honduras. Apparently, therefore, the finding of A. crucians larvae at Bilwas Karma and Puerto Cabezas, Nicaragua, which is reported herewith, is of considerable interest because Puerto Cabezas constitutes the southern limit of the known extension of that species.

Precipitin tests on wild-caught Anopheles walkeri at Reelfoot Lake, Tennessee, demonstrated that this mosquito feeds extensively on cows, horses, and other domestic animals. Although man was not as available as the domestic animals, he was the source of a fair (9 per cent) percentage of blood meals.

Preferential feeding experiments indicated that man is about as attractive to A. walkeri as several species of domestic animals when all are equally available as a source of blood.

A total of 10,412 newly emerged imagines of Anopheles culicifacies were released, from May to October, in a large outdoor screened insectary in 16 batches, varying in size from 100 to 1,793, in order to study length of life of this species. The insects were dusted with printers' ink powder of various colours. Climatic and other conditions prevailing inside the insectary were similar to natural conditions outside.

It was found that the maximum longevity of females varied from 8 to 34 days, being longest later in the season when temperature was slightly lower and relative humidity considerably higher. The longest period a male was observed to live was 8 days.

It was also observed that there was always a very rapid falling off in numbers so that at least 50 per cent of imagines released had died by the third day. Reduction in population was by geometric progression. The probable duration of life of A. culicifacies was only two days, and the average life span only four days. But in each batch there were always one or two individuals that were considerably longer lived than the rest.

Observations over a period of two years in eight comparable villages, four each in malarious Pattukkottai taluk and in the contiguous non-malarious Tanjore delta, in both of which the malaria vector A. culicifacies is found, indicated that the chief factor explaining the phenomenon of anophelism without malaria in the delta was that of density of vector. No morphological, ecological, or behavioristic differences could be found between the A. culicifacies of the two areas. But standardized per man hour collections in human and animal dwellings, and the yield in uniform traps, indicated that the density of A. culicifacies was three to four times greater in the taluk than in the delta, owing to the much greater prevalence and extent of suitable breeding places for this species in the taluk. Intensive agricultural practice seemed to explain the lessened extent of such breeding places in the delta.

These observations bear out previous assumptions that there must be a critical density of a malaria vector species of anopheline, below which no transmission of the disease will occur.

Armigeres and Aedes species of mosquitoes have been infected with, and have transmitted, P. gallinaceum of fowls very easily in our laboratory experiments. But it has not been possible to infect an anopheline, even when fed simultaneously on a fowl whose gametocytes were of a quantity and quality adequate to cause 100 per cent infections in Armigeres and Aedes. With the exception of oocysts in one Culex mimuloides the same difficulty in infecting Culex species has been encountered.

It does not appear that local species of Anopheles or Culex genera are natural hosts for this plasmodium.

A generalized infection in a dog by a fungus proven to be serologically and morphologically identical with Blastomyces dermatitidis, Gilchrist in its various phases, both in the tissues of the host and on artificial media, is reported.

This is believed to be the first record of spontaneous generalized blastomycosis in a dog.

Eleven out of one hundred wild Mus norvegicus rats trapped in Philadelphia were proven to be carriers of Leptospira icterohaemorrhagiae capable of producing symptoms typical of leptospirosis infection in guinea-pigs.

A comparison of the percentage efficiency of the various methods employed to detect infection in the rat materials showed that the darkfield examination of the rat urine was 18.1 per cent; the culture of the urine was 36.6 per cent; the darkfield of the rat kidney emulsion was 18.1 per cent and the culture of the kidney emulsion was 90.0 per cent efficient when compared with the inoculation of albino guinea pigs. Therefore it is suggested that guinea-pig inoculations with rat kidney substance should always be made whenever leptospiral infection is to be investigated in wild rats.

In the course of a study on the origin, distribution and ecology of commensal rats a few facts have become apparent which, I believe, are important enough to be published even before the general account is completed.

1. COMMENSAL RATS OF NORTH AMERICA All the American literature dealing with rats, systematic, ecological, medical, considers three types of these animals: viz. the Norway rat (Rattus norvegicus Berkenhout), the Black rat (Rattus rattus rattus Linnaeus), and the Roof rat (Rattus rattus alexandrinus Geoffroy). This classification, apparently taken from Miller (1924), does not quite cover the facts. The true R. r. alexandrinus Geoffroy is not the animal usually referred to under this name. It has a brown back and grey belly, the two colors gradually passing into one another without any line of demarcation. The other animal, usually referred to as alexandrinus, has a brown back also, but the underside white or lemon yellow and sharply set off from the back.