Marshall Hertig

Natural infections of leptomonad flagellates in Panamanian Phlebotomus sandflies

Abstract In the search for vectors of leishmaniasis in Panama, over 5000 wild-caught Phlebotomus females were dissected and examined for leptomonad flagellates. Natural infections were found in 416 out of a total of 4861 females of six of the seven common man-biting species (gomezi, panamensis, sanguinarius, shannoni, trapidoi, and ylephiletor). A single female longipalpis was also infected. None of 365 females of 13 other species which never or only occasionally bite man was infected. A total of 262 males, including 242 of common man-biting species, was negative. Leptomonad infections were found in sandflies from each of five endemic areas and in two others where human cases have not been reported. The over-all infection rate of the common man-biters was 8.5%, with rates varying from 1.9% for panamensis to 15.4% for trapidoi. During the rainy season, June through December, the over-all rate (10.6%) was much higher than during the dry season (4.1%). The source of these infections in unknown. However, the vertical distribution of species in simultaneous ground-level and tree-platform catches, together with differential infection rates, suggest that the reservoirs may be found among arboreal animals. Leptomonads were always present in the hindgut of infected females. They also occurred occasionally in the posterior part of the midgut (stomach), rarely in the anterior part (cardia), and only twice were they found in the foregut (esophagus and pharynx, respectively). In over 20% of the infections leptomonads occurred in the Malpighian tubules. In culture the morphology of leptomonads from the natural infections is consistent with that of Leishmania. Lesions produced in hamsters by two of the sandfly strains are indistinguishable from those produced by Panamanian human strains. In the sandfly gut the growth pattern and morphology of the leptomonads in natural infections are similar to those of sandflies fed on hamster lesions produced by both Panamanian human strains and a wild-caught sandfly strain.

Experimental Infection of Panamanian Phlebotomus Sandflies with Leishmania.

Abstract Over 800 Phlebotomus sandflies of five species were fed artificially, by the Hertig pipette method, on a mixture of blood and flagellates from cultures of Leishmania braziliensis, s. lat . Flagellate infections of the gut were produced in all five species, with an over-all infection rate of 81%. As in the case of experimental sandfly infections with Old World species of Leishmania causing human disease, growth of the flagellates characteristically took place at the anterior station, i.e., in the cardia and at the proventricular valve, with occasional growth forward into the pharynx. In addition, however, there was usually attachment and growth in the hindgut, particularly in the thin-walled, slightly expanded anterior portion. Attempts to transmit the infection by feeding infected sandflies on spiny rats ( Proechimys ) and a human volunteer were unsuccessful. The inoculation of suspensions of triturated sandflies into spiny rats, suckling white mice, and hamsters produced an infection only once, in a suckling mouse. A small number of sandflies of two species was fed with Leishmania enriettii . Infections of the cardia were produced in one species; the other was completely negative.

Behavior of Leishmania in Panamanian phlebotomine sandflies fed on infected animals.

Abstract Two species of Panamanian sandflies, Lutzomyia sanguinaria and Lu. gomezi, were fed on hamsters infected with various strains of Leishmania. Repeated trials proved that all Panamanian strains of Leishmania braziliensis, whether isolated from man, other mammals, or wild sandflies, produced infections in both sandfly species that were characterized by growth of leptomonad (promastigote) flagellates in the hindgut, especially the “hind triangle,” with or without growth in the midgut. Over 90% of the flies had attached flagellates in the hindgut, and almost half of these had attached flagellates only in the hind triangle. A strain of L. braziliensis from Peruvian espundia reacted similarly in the sandflies. On the other hand, two strains of L. mexicana from Guatemala and British Honduras typically caused midgut infections alone in Lu. sanguinaria and Lu. gomezi. In over 70% of the flies, flagellates were confined to the midgut alone, without even free flagellates in the hindgut. These studies indicate that position of flagellates in the sandfly gut may serve as a reliable taxonomic character for the separation of strains or species of Leishmania.

Immunodiffusion reactions of panamanian Leishmania

Abstract The agar gel diffusion test was utilized in an effort to distinguish five human strains and eight wild-caught Phlebotomus sandfly strains of Panamanian leptomonads plus three human strains from other countries. Concentrated and purified antigens were prepared from cultured organisms by ether extraction, saline extraction, and dialysis. Antisera were prepared in rabbits by using quantified inocula. At least two different immunological groups of human leishmanias have thus been recognized. The members of each group share a number of antigens with each other. There may also be intergroup reactions but they are weaker and usually limited to a single line of precipitate. Within a group, the strains were not limited to a particular geographical location but originated in widely separated sites in the Republic of Panama. Each group contains strains pathogenic for man.

Experimental Inoculation of Panamanian Mammals with Leishmania braziliensis.

Attempts to infect native Panamanian mammals with culture forms of local human strains of Leishmania braziliensis produced cutaneous infections for the first time in the spiny rat (Proechimys semispinosus), white-tailed tree rat (Tylomys panamensis), kinkajou (Potos flavus), and olingo (Bassaricyon gabbii). While infections of hamsters and cotton rats have usually lasted throughout the life of the animals, the parasites disappeared from the spiny rat and tree rat lesions within a month, but persisted in the kinkajous for at least 2 and 3 months. For a number of years we have attempted, at this laboratory, to infect native mammals with human strains of Leishmania braziliensis, sensu lato, in order to discover potential reservoir hosts of leishmaniasis. Early attempts here to infect wild mammals met with failure, but the routine inoculation of golden hamsters and occasional trials with cotton rats from our breeding colonies have shown that these rodents can be readily infected. Although cotton rats of the same species, Sigmodon hispidus, occur in Panama they are not primarily forest animals and so their susceptibility can have only limited significance in the epidemiology of this forest disease. The present paper reports the results of inoculation experiments conducted between June 1963 and September 1964. Lainson and Strangways-Dixon (1964) carried out inoculations of wild animals with Leishmania mexicana in British Honduras. They obtained infections in cotton rats, but of 41 specimens of other wild mammals, representing 11 species, only a single common opossum (Didelphis marsupialis) gave positive results. No lesion was produced at the site of inoculation and the infection was demonstrated only by culture from the liver. MATERIALS AND METHODS There is always the possibility that wild-caught mammals are already immune as a result of natural exposure in the forest. To avoid this difficulty, animals born or reared in the laboratory were used whenever available. In 1963 the breeding of the Received for publication 2 April 1965. * The work reported here was supported in part by a research grant (AI-01251) from the National Institute of Allergy and Infectious Diseases, NIH, USPHS. 842 arboreal white-tailed rat (Tylomys panamensis) was initiated at this laboratory. Although the average litter size was found to be only two, and the gestation period about 35 days, it has now been possible to build up the colony to about 130 individuals. Most of the Tylomys used in this study were laboratory-bred from wild-caught parents. The spiny rats (Proechimys semispinosus) were born in the laboratory from wild-caught gravid females. The woolly opossums (Caluromys derbianus) were laboratory-reared, but were brought into the animal room in the marsupia of wildcaught females. The cotton rats (Sigmodon hispidus) were from our colony established several years ago with stock from commercial sources in the U. S. The other animal species were wildcaught. Most of the live-trapped mammals were obtained near the town of Achiote, Province of Colon, Panama, where human cutaneous leishmaniasis has been known to be endemic for many years. The two strains of Leishmania used in the present study were isolated in Senekjies modification of NNN culture medium from the cutaneous lesions of patients. The first of these isolates (Mstrain) was obtained in December 1960 from a patient having multiple lesions. The patient was a 48-year-old male who came to the laboratory for treatment. He had spent most of his life near Concepcion, in Chiriqui Province, but had developed the lesions while on the Changuinola River in Bocas del Toro Province, a known endemic area. The patient reported that the first lesion had started on the back of his hand in October 1960, and that about 3 weeks later others began to appear. When seen at the laboratory, the patient had an ulcer of from 1 to 3 cm in diameter in each of the following locations: hand, arm, tip of nose, thigh, neck, and left ear. This strain has been maintained in culture and in hamsters. The other strain used in this study (VH-strain) is of more recent origin. In April 1964 one of our fieldmen developed a large lesion on his upper left arm while engaged in trapping mammals in the Achiote area. This man has worked in the field for the Gorgas Memorial Laboratory for 28 years, and had been exposed in endemic areas many times w thout previously contracting leishmaniasis. Treatment was initiated as soon as the condition This content downloaded from 157.55.39.166 on Wed, 24 May 2017 20:53:57 UTC All use subject to http://about.jstor.org/terms THATCHER ET AL.-EXPERIMENTAL LEISHMANIASIS IN PANAMANIAN MAMMALS 843 TABLE I. Inoculations of Panamanian mammals with two human strains of L. braziliensis.