Charles W. Young

The development of flagellates in Chinese sandflies (phlebotomus) fed on hamsters infected with Leishmania donovani.

Sandflies (Phlebotomus) have assumed a particularly important position in the study of the leishmaniases since Knowles, Napier and Smith 1 reported the appearance of herpetomonad flagellates in a large proportion of Phlebotomus argentipes fed on kala azar patients in Calcutta. The present paper is a report of certain phases of studies undertaken on the sandflies of North China as possible transmitting agents of kala azar. Three species of Phlebotomus are known to us, namely, Phlebotomus major var. chinensis Newstead, and two unidentified species which we have designated Phlebotomus “B” and “C”. Phlebotomus “B” is apparently the unnamed species mentioned by Newstead. 2 These three species occur in markedly variable proportions in several regions near Peking and Hsüchowfu, Kiangsu. In these studies (1) sandflies captured in houses of kala azar patients and elsewhere have been examined for flagellates. (2) Sandflies reared in the laboratory have been fed on kala azar patients and on hamsters heavily infected with Leishmania donovani. In the attempt to transmit kala azar these sandflies have been refed upon tested negative hamsters, and a certain number have been inoculated into other hamsters. As many as possible of such sandflies were examined for the presence of flagellates. A total of over 250 hamsters used in these transmission studies were all negative by liver puncture from 81 to 137 days after the experiments, but final results will be reported from autopsy findings. It is desired to report at this itme (1) a technique of rearing sandflies and of feeding them on hamsters, with particular reference to the feeding of these insects a second time, a feature hitherto little studied, and (2) the results of examination for herpetomonad flagellates. Technique. The method of rearing sandflies, which will be reported in detail in a separate paper, is a modification of the methods of Waterston 3 and Srnithb 4 The breeding vessels are porous earthen pots with a thin lining of plaster of Paris.

Peripheral Lesions produced by L. donovani and Allied Leishmaniae.

Gonder, 1 Laveran, 2 , 3 and Sergent 4 have reported peculiar lesions of the extremities, tail, scrotum, nose and ears in white mice inoculated intraperitoneally with recently isolated cultures of L. tropica. Sergent 4 was unable to produce such lesions with cultures beyond the 50th cultural passage. Bramachari 5 and others in Calcutta have reported cases of Kala Azar treated with antimony and apparently cured, in whom there developed later, nodules in the skin of the face, upper extremities and trunk, in which leishmaniae were found and from which they were cultivated. The spleen, liver and blood stream in these cases were free from leishmaniae. Bramachari has called this complex “dermal leishmanoid”. Acton and Knowles 6 reported a patient, diagnosed clinically as xanthoma tuberosum multiplex. A leishmania was cultivated from the lesions. The present authors worked, in part, with cultures obtained from Drs. Nicolle and Anderson of Tunis, who have had them under continuous artificial cultivation for varying periods up to nearly 15 years. These cultures comprised L. donovani (L. infantum), strains KA and Sh, L. canis (“kala azar canin”) strains x and X, and L. tarentolae (“leptomonas de gecko”) from the gecko. All of these strains originally produced visceral lesions only. When these cultures were inoculated intraperitoneally into Chinese striped hamsters (Cricetulus griseus), the infections were visceral at first, with enlarged spleen and liver and with leishmania fairly abundant in the smears from the spleen, liver, bone marrow and heart blood.

Kala Azar Transmission Experiments with Chinese Sandflies (Phlebotomus).

We reported 1 the development of herpetomonad flagellates in Chinese sandflies fed on hamsters infected with kala azar. In the present paper are summarized the results of various kala azar transmission experiments performed in 1925 and 1926. The three species of sandflies, for the determination of which we are indebted to Professor R. Newstead, are: Phlebotomus major var. chinensis Newstead; P. sergenti var. Newstead (Phlebotomus “C” of our previous report), identical with an unnamed variety described from Mesopotamia 2 ; P. perturbans var. Patton 3 (Phlebotomus “B”). This latter species we have never fed on man or other mammals in the laboratory, but it readily engorges on toads. In the following experiments we tested the hypothesis that Phlebotomus sandflies becoming infected by sucking the blood of kala azar patients are then able to transmit the disease to other persons by their bites. The first was fed upon heavily infected hamsters. Later they were fed upon tested negative hamsters. As P. sergenti became infected with Leishmania only rarely, following “natural” feeds, as contrasted with the very large proportion of infected P. major, a technique described elsewhere 4 was devised for feeding sandflies artificially with rich suspensions of Leishman-Donovan bodies. The apparatus consists essentially of a cork vise for immobilizing the sanctfly and an adjustable holder for the feeding pipette, the end of which is fitted over the insects stylets. Freshly defibrinated rabbit blood was centrifuged and the serum removed. Spleen tissue from a hamster heavily infected with Leishmania was ground up in this serum. A volume of spleen-serum suspension equal to that of the serum, originally removed, was then added to the rabbit corpuscles. The sandflies engorged readily. Infections with Leishmania were produced in all three species. Those for P. perturbans are not reported here, since feeding experiments on mammals have not proved possible with this species.

Experimental kala-azar in a hamster.

Summary and Conclusions A Chinese hamster (Cricetulus griseus, M.-Edw.) is very susceptible to inoculation with the aflagellate form of Leishmaniadonovani when the organisms are injected intraperitoneally. It is also susceptible to intraplleural inoculation. It is sometimes but not uniformly infected by subcutaneous inoculation. The two feeding experiments were negative.

A search for field and house rodents naturally infected with kala-azar.

The striped hamster (Cricetulus griseus) has been shown to be very susceptible to infection with kala-azar. 1 Its southern range corresponds, so far as it has been studied, with the distribution of kala-azar in China. These two facts together with other observations on the epidemiology of the disease, suggested the possibility that this hamster or some other field rodent may be a reservoir of Leishmania donovani. Assuming such a source, man might be infected in his dwelling directly from the animal or indirectly by means of some domestic rodent (rat or mouse) which had in turn become infected from an invading field rodent. In either case the probable vector or intermediate host would be some rodent ectoparasite. With this rodent-ectoparasite hypothesis in mind we have: (1) searched for hamsters and other field and house rodents naturally infected with kala-azar; (2) tested for susceptibility to infection with Leishmania donovani the common field and house rodents from an endemic area; and (3) studied the natural ectoparasites of these rodents and their capabilities as transmitters of the disease. The present paper is a brief statement of the results of the first of these investigations. The animals recorded comprise (1) those killed and examined primarily for the purpose of searching for naturally infected animals; (2) those that died in the laboratory before they had been submitted to any experimental procedure and (3) such stock rodents as were accidentally killed by liver puncture. From this study were excluded (1) all rodents that had received any injection or had been submitted to any manipulation other than liver punture; (2) all carcasses that showed decomposition of the organs; and (3) all animals regarding whose exact origin and subsequent history there was any doubt.

Susceptibility of Field, House and Laboratory Rodents to Infection with Leishmania donovani.

Conclusions 1. The giant hamster (Cricetulus triton), a vole (Microtus sp.) and the Chinese house mouse (Mus wagneri) are markedly susceptible to infection with Leishmania donovani. These showed no tendency toward spontaneous recovery during the periods covered by the present studies. 2. The house rat (Mus ruttus) white rat (Mus novegicus (vel decunzanus) albinus) and white mouse (Mus musculus albinus) show infections of only moderate intensity and seem to exhibit a tendency to recover spontaneously. However none of the series was long enough to show undoubted and complete recovery.

The diagnosis of kala-azar by blood culture

Summary 1. Human red cells and serum are unfavorable to the growth of Leishmania donovani. 2. A method is given for removing the red cells and serum from blood before planting. 3. By this method blood cultures have been obtained from nine out of ten samples of blood from five patients, some of them after considerable antimony treatment. 4. A modified “N.N.N.” medium is suggested. 5. On this medium the Leishman-Donovan bodies from spleen punctures or peripheral blood develop into flagellates at all hydrogen-ion concentrations tested, i.e., between PH 6.8 and PH 8.2 and the flagellates grow at least to PH 9.0. 6. Cultures on this modified medium show post-flagellate forms and perhaps Cornwalls “thick tails.”

Attempts to transmit kala azar by means of bedbugs (cimex sp.).

Summary Attempts to transmit kala azar from heavily infected hamsters to highly susceptible negative hamsters by means of Cimsx lectularius and Cirrzex pipistrelli have been unsuccessful.

Attempts to transmit kala azar by means of rodent lice, haematopinus sp.

Summary (1) No development of Leishmania donovani in the two species of hamster lice studied was demonstrated, but these organisms occasionally survive for a short period in the striped-hamster louse. (2) Attempts to transmit kala-azar from hamster to hamster by means of hamster lice were completely unsuccessful.

Leishmania donovani in the Peripheral Blood.

Donovan 1 who found Leishmania donovani first in the living patient also discovered the parasite in smears from the peripheral blood. Many have advocated the examination stained blood smears for diagnosis of the disease (Patton, 2 Marshall, 3 Cannata, 4 Knowles 5 ) but all agree that the organism is present in small numbers and that many slides must be carefully studied. Spleen smears obtained by aspiration has been the usual method for diagnosis. As the bleeding time is prolonged in all advanced cases this procedure is not devoid of danger (Donovan, 1 Wylie, 6 Knowles, 7 Rogers, 8 Bramachari 9 ). Blood culture has been suggested as a substitute but results have been inconstant (Mayer and Werner, 10 Wenyon, 11 Row, 12 Korke, 13 Knowles 5 ). The last named author made 128 cultures from 34 patients with two positive cultures from one patient. At first Rogers merely citrated the patients blood but Nicolle was much more successful with his “N.N.N.” medium, a rabbit blood agar. The experiments of Rogers 15 and of Cornwall and LaFrenais 14 showed that culture media made with human blood were not favorable to the growth of Leishmania donovani. Two series of experiments were made using salt agar as in N.N.N. medium but with washed red cells and serum, both unheated and heated at 56 C° for one-half hour, from man, horse, sheep and rabbit. Human red cells were somewhat inhibitory; human serum much more so. Rabbit cells and serum were less unfavorable to growth but were inferior to whole defibinated rabbit blood. In action the cells and serum of the horse and sheep lay between those of man and the rabbit. As a result of these experiments, a technic for blood culture was devised as follows: 10 c.c. of blood was drawn from a vein into 2 c.c. of 1 per cent. citrated Lockes solution in a syringe and immediately expelled into 50-70 c.c. of the same fluid in a flask.