States-Japan Cooperative Medical Science Program Research Articles

ハンセン病研究と日米医学協力計画

ハンセン病研究と日米医学協力計画

20th International Conference on Emerging Infectious Diseases in the Pacific Rim Organized by the United States-Japan Cooperative Medical Sciences Program (USJCMSP).

The 20th International Conference on Emerging Infectious Diseases in the Pacific Rim to3ok place in Shenzhen, China on January 8–9, 2018 followed by meetings of the acquired immunodeficiency syndrome (AIDS)/immunology, acute respiratory infections, cancer, hepatitis, and viral diseases panels on January 10–11. The conference was organized as part of the United States-Japan Cooperative Medical Sciences Program (USJCMSP) by the Japan Agency for Medical Research and Development (AMED) and the U.S. National Institutes of Health (NIH) and was locally hosted by the Shenzhen Third People’s Hospital and the Chinese Academy of Sciences (CAS) Institute of Microbiology. The conference provides the basis for networking and fostering of collaboration opportunities between researchers in Southeast Asia and the United States based on the scientific and interactive platform of the USJCMSP and takes place in the region on an annual basis. This report summarizes the discussions and conclusions from the conference.

Reframing Critical Needs in Vector Biology and Management of Vector-Borne Disease

Reframing Critical Needs in Vector Biology and Management of Vector-Borne Disease

The United States-Japan cooperative medical sciences program conference on emerging diseases

The United States-Japan cooperative medical sciences program conference on emerging diseases

A Technique for Selecting Uniform Samples of Schistosoma mansoni Based on Egg Production

cit.); and (3) infections in which the sex ratio was fairly well-balanced (1 + 0.31). The data presented in Table II indicate that their mean parasite fecundity value was indeed lower (768 + 137 eggs per day per female). The difference between the means of the balanced sex ratio group (1,107 ? 258) and the imbalanced sex ratio group (768 + 137) is highly significant (P < 0.02). Although imbalanced sex ratios were developed in different ways in the murine study (unisexual super-infection) and in the present baboon study (primary infection imbalance owing to undetermined causes), the results were similar, suggesting that a sex ratio effect on egg production occurs in simian S. mansoni infection as well. The present and Cheever and Duvall's (1974, loc. cit.) estimates of S. mansoni egg production rates are two or three times higher than the earlier estimates, and strongly support the notion of a higher reproductive potential, and therefore a higher pathogenic potential for this organism than was previously assumed. Further precision is currently being sought through our attempts to experimentally determine the half-lives of S. mansoni eggs in the tissues of baboons with various degrees and durations of infection and with various degrees of immunity. This study was supported by the United StatesJapan Cooperative Medical Sciences Program administered by the National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Public Health Service, Department of Health and Human Services (Grant Al 18906).

On the Longevity of Larvae of the Hookworm Uncinaria lucasi in Tissues of Northern Fur Seals (Callorhinus ursinus)

Adult worm recoveries are shown in Table I. One male neonate mouse given 80 larvae developed a patent infection at 60 days and remained patent, although at low levels (2-5 microfilariae/100 ,l of blood), until killed at 140 days. All other mice failed to develop a microfilaremia. Six of the neonate mice infected with 40 larvae harbored adult worms. All mice infected as neonates with 80 larvae had adult worm burdens. None of the control mice infected at 8 wk of age harbored adults or microfilariae at necropsy. Approximately 77% of the adults were recovered from the peritoneal cavity. Worms were also found in the heart, lungs, reproductive organs, and thoracic cavity of some mice. There was a preponderance of female worms recovered (1 329, 676); the majority of these female worms, however, were recovered from the peritoneal cavities of only two mice. Male and female neonate mice appeared to be equally susceptible to infection. It is perhaps noteworthy that the only mouse that developed a patent infection had adult worms in the thoracic cavity. Although the evidence is based on only one animal, it is possible that the thoracic cavity may be a better site for worm development than the peritoneal cavity, lungs, heart, or reproductive organs. Most worms appeared viable, although a few partially calcified worms were found. The size of the adult worms recovered 140 days after infection was consistently smaller than those found in either cats (Schacher, 1962, J. Parasitol. 48: 693-706) or jirds (Ash and Riley, 1970, J. Parasitol. 56: 962-968); in the mice that were infected as neonates, female worms averaged 30.8 mm and male worms averaged 12.8 mm in length. The successful establishment of Brugia pahangi infection in neonate mice opens up the possibility of the ready availability of murine filarial infections. Experiments should be done to delineate the best protocol for infecting animals and the subsequent development of adult worms and microfilaremias. Mice harboring adult worms should be studied to detail the immunologic parameters that render neonate mice susceptible to infection. This work was supported by a research grant, No. AI-10250, from the United States-Japan Cooperative Medical Science Program. We are grateful to Virginia Hansen for editorial support.

Observations of Aedes albopictus cell cultures persistently infected with Sindbis virus.

Annals of the New York Academy of SciencesVolume 266, Issue 1 p. 214-231 OBSERVATIONS ON AEDES ALBOPICTUS CELL CULTURES PERSISTENTLY INFECTED WITH SINDBIS VIRUS* Victor Stollar, Victor Stollar Department of Microbiology College of Medicine and Dentistry of New Jersey Rutgers Medical School Piscataway, New Jersey 08854Search for more papers by this authorThomas E. Shenk, Thomas E. Shenk Department of Microbiology College of Medicine and Dentistry of New Jersey Rutgers Medical School Piscataway, New Jersey 08854 Department of Biochemistry, Stanford University School of Medicine, Stanford University Medical Center, Stanford, Calif. 94305.Search for more papers by this authorRose Koo, Rose Koo Department of Microbiology College of Medicine and Dentistry of New Jersey Rutgers Medical School Piscataway, New Jersey 08854Search for more papers by this authorAkira Igarashi, Akira Igarashi Department of Microbiology College of Medicine and Dentistry of New Jersey Rutgers Medical School Piscataway, New Jersey 08854Search for more papers by this authorR. Walter Schlesinger, R. Walter Schlesinger Department of Microbiology College of Medicine and Dentistry of New Jersey Rutgers Medical School Piscataway, New Jersey 08854Search for more papers by this author Victor Stollar, Victor Stollar Department of Microbiology College of Medicine and Dentistry of New Jersey Rutgers Medical School Piscataway, New Jersey 08854Search for more papers by this authorThomas E. Shenk, Thomas E. Shenk Department of Microbiology College of Medicine and Dentistry of New Jersey Rutgers Medical School Piscataway, New Jersey 08854 Department of Biochemistry, Stanford University School of Medicine, Stanford University Medical Center, Stanford, Calif. 94305.Search for more papers by this authorRose Koo, Rose Koo Department of Microbiology College of Medicine and Dentistry of New Jersey Rutgers Medical School Piscataway, New Jersey 08854Search for more papers by this authorAkira Igarashi, Akira Igarashi Department of Microbiology College of Medicine and Dentistry of New Jersey Rutgers Medical School Piscataway, New Jersey 08854Search for more papers by this authorR. Walter Schlesinger, R. Walter Schlesinger Department of Microbiology College of Medicine and Dentistry of New Jersey Rutgers Medical School Piscataway, New Jersey 08854Search for more papers by this author First published: November 1975 https://doi.org/10.1111/j.1749-6632.1975.tb35103.xCitations: 24 * Supported by Grant GB37707 from the National Science Foundation, by Grant AI-11290 from the National Institute of Allergy and Infectious Diseases, and by the United States-Japan Cooperative Medical Science Program through United States Public Health Service Grant AI-05920. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume266, Issue1Pathobiology of Invertebrate Vectors of DiseaseNovember 1975Pages 214-231 RelatedInformation

Autoradiographic Studies on the Reproductive System of Philophthalmus hegeneri Penner and Fried, 1963

Adult Philophthalmus hegeneri exposed in vitro to 3H-thymidine showed incorporation of the isotope on autoradiograms over nuclei of actively dividing cells of the testes, ovary, vitellaria, and developing miracidia within eggs. Autoradiograms of worms exposed for 1 hr to 3H-tyrosine showed heavy labeling over the ovary, vitelline cells, and the testes. Timing studies utilizing transplanted thymidine-labeled worms indicated it takes 12 days for oogonial cells to become labeled, migrate through the ovary, and be incorporated into eggs. It took 96 hr for labeled vitelline cells to reach the vitelline reservoir. Spermatogonia labeled in the initial 6-hr exposure took 48 hr to become primary spermatocytes, 72 hr to become secondary spermatocytes, 96 hr to become spermatids, 120 hr to become mature sperm in bundles, and 156 hr to reach the seminal vesicle. Studies with tyrosine-labeled worms transplanted to the host for 5 days indicated adults taken from multiple infections will not self-inseminate when isolated. Similar worms transplanted with unlabeled worms exclusively cross-inseminate. In a study on an eye fluke of birds, Philophthalmus megalurus, utilizing techniques of radioisotopic labeling, autoradiography, and transplantation, Nollen (1968a) was able to study several aspects of reproductive physiology and behavior. Timing of stages of gametogenesis and migration of vitelline cells as well as patterns of sperm transfer in single and multiple infections were determined in this study.Nollen found these flukes can selfinseminate in isolation but preferentially crossinseminate in multiple infections. Philophthalmus hegeneri, an eye fluke of shore birds in Florida, differs in several ways from P. megalurus. Among these are the apparent lack of self-insemination when adult P. hegeneri are grown in isolation (Fried, 1962). The ability of the snail intermediate host, Batillaria minima, to survive long periods out of the water makes it ideal for shipping. Thus we have been able to raise adult worms in chicks from larval stages shed by snails collected in Florida. The techniques utilized by Nollen (1968b) were applied to a study of P. hegeneri to see if species differences are apparent in reproductive physiology and behavior when compared to P. megalurus. MATERIALS AND METHODS Philophthalmus hegeneri was obtained from naturally infected Batillaria minima collected from Received for publication 2 March 1973. * Supported by the United States-Japan Cooperative Medical Sciences Program administered by NIAID: Grant Al 09330. Clearwater Harbor, Largo, Florida. Cercariae shed from snails encysted in finger bowls and were stored in aerated seawater not longer than 4 days before use. Metacercariae were induced to excyst by replacing seawater with warm (40 C) saline (0.85%), and day-old chicks (Big H Foods, Fairbury, Ill.) were infected by pipetting the metacercariae directly into the orbit. Worms were removed from the conjunctival sacs of chicks after 35 to 45 days of growth and exposed at 38 to 39 C in small stender dishes to tritiated compounds (Amersham/Searle Inc.) diluted with HedonFleig's saline. The radioisotopes used were thymidine (methyl-3H; 22.4 Ci/mmole) at 75 ,uCi/ml and L-tyrosine (-3,5-T; 1 Ci/mmole) at 100 iCi/ml. Worms were exposed to 3H-thymidine for periods of 6 hr or more to determine the time needed for optimum labeling of the cells of the reproductive system. Similar exposures were carried out with 3H-tyrosine to determine areas of incorporation in the tissues of P. hegeneri. After exposure, worms were either killed immediately in alcohol-formalin-acetic acid (AFA) or returned to the conjunctival sacs of uninfected chicks for varying periods of time. Techniques for autoradiography and transplantation of the exposed worms are described by Nollen (1968b).

Foleyella flexicauda sp. n. (Nematoda: Filarioidea) from Rana catesbeiana in New Jersey, with a Review of the Genus and Erection of Two New Subgenera

Foleyella flexicauda sp. n. from the mesentery and body cavity of Rana catesbeiana in New Jersey most resembles F. brachyoptera Wehr and Causey, 1939, and F. americana Walton, 1929. Males of the new species differ from those of F. brachyoptera by having a longer esophagus and caudal alae which terminate behind rather than at the cloaca, while females are shorter, have a longer tail and a more anteriorly placed vulva. Males have longer spicules and a smaller spicule ratio than those of F. americana; caudal alae of the latter extend to the tip of the tail. F. americana has 4 pairs of pedunculate preanal papillae, lacking in the new species. In F. flexicauda sp. n. the vulva is always esophageal in position, in F. americana it is postesophageal. The genus is reviewed and 2 new subgenera are erected which coincide with host types. Foleyella (Foleyella) subgen. n. comprises 5 species parasitic in agamid and chamaeleontid lizards. Its type is F. (F.) candezei (Fraipont, 1882) Seurat, 1917. Foleyella (Waltonia) subgen. n. comprises 8 species found in Salientia; its type is F. (W.) duboisi (Gedoelst, 1916) Yorke and Maplestone, 1926. Subgenera are morphologically differentiated on the presence (Waltonia) or absence (Foleyella) of conspicuous longitudinal alae, the presence of stomal support structures (Waltonia), and smooth (Foleyella) vs. tuberculate (Waltonia) ventral precloacal cuticle. The microfilarial sheath is usually inflexible and spindle-shaped or ovoid in F. (Foleyella), flexible and appressed in F. (Waltonia). Infective larvae of F. (Waltonia) spp. have an esophagus one-half or more the body length; this is shorter in F. (Foleyella) spp. F. convollta (Molin, 1858), F. leiperi (Railliet, 1916), F. scalaris Travassos, 1929, and F. vellardi Travassos, 1929, are considered undiagnosable pending restudy. F. helvetica Kreis, 1934, is synonymized with Icosiella neglecta (Diesing, 1851) Seurat, 1917. The genus Foleyella Seurat, 1917, mostly comprises filariae of amphibia, but has several representatives found in agamid and chamaeleontid lizards. The genus has been reviewed by Witenberg and Gerichter (1944), Skrjabin and Shikhobalova (1948), L6pez-Neyra (1956), and Sonin (1968). Brygoo (1963) and Schacher and Khalil (1967) reviewed reptilian forms; the latter study reinstated one species and described yet another, bringing to five the number found in lizards. Sonin's (1968) review accepted as valid eight species from amphibia: Foleyella americana Walton, 1929; F. brachyoptera Wehr and Causey, 1939; F. convoluta (Molin, 1858) Travassos, 1929; F. dolichoptera Wehr and Received for publication 19 January 1973. * This investigation was supported in part by the United States-Japan Cooperative Medical Science Program administered by the NIAID, NIfI, Department of Health, Education and Welfare, Grant 2R 22 AI-07770. t Division of Infectious and Tropical Diseases, School of Public Health, University of California Los Angeles, Los Angeles, California 90024. t Department of Entomology and Economic Zoology, Rutgers University, New Brunswick, New Jersey 08903. Cause , 1939; F. duboisi (Gedoelst, 1916) Yorke and Maplestone, 1926; F. leiperi (Railliet, 1916) Yorke and Maplestone, 1926; F. ranae Walton, 1929, and F. vellardi Travassos, 1929. He rejected F. bouillezi (= Filaria sp. of Bouilliez, 1916) Witenberg and Gerichter, 1944, as a synonym of F. leiperi, and reinstated Foleyellides striatus (Ochoterena and Caballero, 1932) Caballero, 1935, which Witenberg and Gerichter (1944) had placed in Foleyella. To Sonin's list must be added F. confusa of Schmidt and Kuntz (1969). The present study describes a new species of Foleyella, first discovered by Crans (1969), and differentiates it from the four species previously recorded from amphibia in the United States. The genus is reviewed and two new subgenera are erected. MATERIALS AND METHODS Adult worms, recovered from infected Rana catesbeiana as described by Crans (1969), were heat-killed in saline and transferred immediately to AFA at 60 C. Subsequent treatment and study was as described by Schacher and Khalil (1967). Microfilariae were studied in air-dried smears of fr g blood presoaked in Earle's solution (Spinner modification) and immediately fixed 1/ hr in a freshly prepared 3:1 mixture of glacial acetic acid

Occurrence of Mouse α 2 -Macroglobulin Antigenic Determinants on Schistosoma mansoni Adults, with Evidence on Their Nature

Rabbits immunized with washed, homogenized adult Schistosoma mansoni worms recovered from mice (mouse worms) developed precipitins which reacted with mouse yG2 and mouse ca-macroglobulin (a2M). Both antibodies could be absorbed from the rabbit antisera with mouse worms or mouse serum. Only the anti-o_M could be absorbed with monkey worms. Rhesus monkeys immunized with purified mouse a2M and subsequently challenged with mouse worms surgically implanted in their hepatic portal systems did not show an increased ability to destroy the mouse worms. Direct identification of mouse a2M antigenic determinants on the worms' surfaces was made by mixed agglutination modified for use with mouse o2M-coated red cells. Both male and female worms possess the determinants, with male worms apparently having more. As demonstrated by mixed agglutination, mouse worms, monkey worms, and mouse worms recovered after 1 month in rhesus monkeys were all alike in possessing mouse acM antigenic determinants. Because of the lack of cross-reactivity between primate and murine c2-macroglobulins and because mouse a2M antigenic determinants are also found on monkey worms, it is concluded that these shared host-parasite antigenic determinants are constitutive determinants synthesized by the schistosome. Interest in or hostlike antigenic determinants in Schistosoma mansoni (Capron et al., 1965, 1968; Damian, 1964, 1967) has accelerated with the evidence from Smithers' laboratory that schistosomes acquire host antigens, both in vivo (Smithers et al., 1969; Clegg et al., 1970) and in vitro (Clegg et al., 1971). Various theories have been advanced to explain host-parasite antigen sharing. Antigen mimicry theories, e.g., those suggesting that there are constitutive hostlike determinants synthesized by parasites (Sprent, 1962; Dineen, 1963; Damian, 1964), are not supported by the evidence that certain host antigens, disclosed by heterologous transfer experiments, are evanescent (Smithers et al., 1969). Rather, this evidence favors equally an antigen incorporation theory (Smithers et al., 1968) and the theory of host-specific antigen synthesis by the parasite through a host-triggered induction process (Capron et al., 1968). The present investigations of host-parasite antigen sharing were stimulated by the observation of one of us (W.J.H.) that rabbits imReceived for publication 11 April 1972. * This investigation was supported by the United States-Japan Cooperative Medical Science Program administered by the NIAID, NIH, Department of Health, Education and Welfare, Grant 5 R22 AI08398. munized with homogenized adult schistosomes recovered from mice (mouse worms) developed precipitins reactive with a mouse serum aglobulin. This protein was subsequently characterized and identified as x2-macroglobulin (a2M) by Greene et al. (1971). When first noted, it was thought that the protein represented the acquired mouse determinants that Smithers et al. (1969) demonstrated by surgically transferring mouse worms into antimouse monkeys. The research reported herein was designed to test this hypothesis and to provide further information on the nature of the determinants in question. MATERIALS AND METHODS A Kenyan strain of Schistosoma mansoni was used. It was originally isolated from a patient and subsequently passaged through mice and hamsters, but since establishment in our laboratory in 1968, the parasite has been maintained exclusively by passage through Biomphalaria pfeifferi and savanna baboons (Papio cynocephalus). Mice and rhesus monkeys were infected from the maintenance stock only as needed for specific experiments. Adult worms were recovered from mice and rhesus monkeys by modifications of the perf-o-suction perfusion method (Radke et al., 1961). Worms to be used for immunogen, antiserum absorptions, surgical transfers, or in vitro tests were transferred from the perfusion fluid to phosphate-buffered (pH 7.4, 0.01 M) saline (PBS) and teased free of any adhering debris under a stereoscopic micro-

Scanning Electron Microscopy of Integumental Surface of Schistosoma mansoni

The integumental surface of adult S. mansoni was studied by scanning electron microscopy at 50 to 10,000 magnifications. Many spines of variable size cover the inner surface of the oral sucker of the male and extend to the pharyngeal opening. Fewer and smaller globular spines cover the inner surface of the ventral sucker. Bosses of variable size with variable numbers (50 to 250) of spines occur on the greater part of the body surface but not in the gynecophoral canal. The posterior end of the male is devoid of bosses, but spines are freely distributed over the surface. The dorsal body surface is also characterized by papillalike integumental elevations of irregular size, shape, and distribution. These may be associated with clumps of small spines, and in some instances possess a cavity or crater. Larger spines protrude from some of the cavities. The female is free of bosses, but there is a heavy covering of anteriorly directed spines on the posterior part of the body. The structure of the integument of the schistosomes, particularly Schistosoma mansoni, has been investigated employing ultrathin sections suitable for examination in transmission electron microscopy (TEM) (Senft et al., 1961; Morris and Threadgold, 1968; Silk et al., 1969; Smith et al., 1969). These studies have indicated that the integument is a syncytium made up of a surface layer recognized as a cytoplasmic sheath or matrix, connected by cellular extensions to cell bodies (nuclei) buried in the parenchyma. Additionally, there are ultrastructural objects including mitochondria, cuticular channels, vesicles, and discoid granules. Golgi bodies, endoplasmic reticulum, and ribosomes are absent. Contrasted with the abundance of information dealing with subsurface integument, there is a paucity of observations, at comparable magnifications, made on the surface of the schistosome. This is understandable since there was no readily available means of examining the ultrastructure of surfaces other than by utilizing the incalculably small amount of information which could be derived Received for publication 23 March 1972. *Investigation supported by the United StatesJapan Cooperative Medical Science Program administered by the NIAID, NIH, Department of Health, Education, and Welfare, under Grant No. 5 R 22 AI-08207. t Department of Pathology and Laboratories, Nassau County Medical Center, East Meadow, New York 11554. t Division of Microbiology and Infectious Diseases, Southwest Foundation for Research and Education, San Antonio, Texas 78228. from nonsequential ultrathin TEM sections which pass through the surface. The roughness of the surface of the male S. mansoni and, conversely, the smoothness of the female first was recognized employing light microscopy. By this means it was possible to identify many elevated areas which were designated as tubercles covered with hairs. With the development of TEM came a change in nomenclature, i.e., the tubercles became bosses and the hairs became spines. Additionally, TEM occasionally revealed sections through such surface structures as openings of canaliculi and pores of various types. The present report is concerned with the surface microtopography of the adult S. mansoni as visualized with the scanning electron microcope (SEM). The SEM, with its great depth of field, i.e., at least 200 times that of a light microscope, and its resolution of 200 A or better, provides a means for determining the fine topography of body surfaces. Furthermore, an opportunity is afforded to correlate external features with those internal structures revealed by TEM. Other studies dealing with the ultrastructure of the schistosome surface are in progress and involve the use of specimens of known ages from 1 day to 27 weeks, i.e., from the schistosomule to the mature adults. By this means the maturation of surface structures may be correlated with time. To date, the SEM has been used by Hockley (1968), Johnson and Moriearty (1969), and Robson and Erasmus (1970) to study the surface features of the cercariae of S. mansoni, and by

On the reported occurrence of an ecdysone-like steroid in the freshwater snail, Biomphalaria glabrata (Pulmonata; Basommatophora), intermediate host of Schistosoma mansoni.

Attempts to repeat the isolation of an ecdysone-like steroid from Biomphalaria glabrata revealed that some of the manipulations described by Muftic (1969) were physically impossible to repeat. With modified procedures no ecdysone-like steroid was found. The original publication gives scant attention to certain experimental details. Some sections of the text bear very close resemblance to earlier publications by different authors. The amount of substance claimed to have been isolated, and the long survival period claimed for Schistosoma mansoni larvae in vitro, are claims which, amongst others, seem highly unlikely a priori. All attempts to contact Muftic have proved to be futile. It is suggested that the paper is, at least in part, erroneous and that any future reference to it should clearly indicate its suspected invalidity.Despite these results, it is considered likely that future investigations will demonstrate physiological roles for steroids in snails.I wish to acknowledge the stimulating communications of Drs E. L. Hansen, M. Voge, R. E. Thorson, C. S. Richards, J. N. Kaplanis, G. Nace, R. Monroe, and of E. Chernin, who first brought this matter to my attention. Theanalytical steroid work could not have been done nor interpreted without the considerable time and effort willingly given by Drs F. Kohen and R. Counsell of the University of Michigan College of Pharmacy. My snail populations were supplemented with snails from the tanks of Mrs B. Caveny. Technical help was given by E. Perisho, G. Lindsay and P. LoVerde. Financial support was kindly provided by Dr J. B. Burch. This investigation was supported in part by the United States-Japan Cooperative Medical Science Program administered by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health, Department of Health, Education and Welfare, grant N.I.A.I.D. AI-7364, and in part by NSF grant GB-29315. Improvements were made to the first draft of the manuscript after it was critically read by Drs E. L. Hansen, F. Kohen, M. Voge, E. Chernin, R. E. Thorson and J. N. Kaplanis.

Distribution of Developing and Mature Brugia malayi in Rhesus Monkeys and in the Jird, Meriones unguiculatus, after a Single Infection

Ten rhesus monkeys (Macaca mulatta) and 20 jirds (Meriones unguiculatus) were each given a single inoculation of subperiodic Brugia malayi infective larvae in the rear foot by a puncture wound technique. The monkeys were killed after a period varying from 5 days to 25 weeks and the jirds from 2 to 32 weeks following inoculation. The individual recovery of worms from monkeys ranged from 0% to 40% with an average of 12%. Out of an overall total of 121 worms recovered from the 10 monkeys, 78 were found in the lymph nodes of the inguinal area, and 21 in the lymph vessels of the infected leg distal to the popliteal node. In jirds the individual recovery varied from 3% to 40% of the administered inoculum with an overall average of 207%; up to 6 weeks following infection, the majority were found in the lymph vessels distal to the popliteal node; from 7 to 32 weeks following infection, the majority were located in the heart and lungs with some worms recovered from the testes. This study confirms the production of patent infections of Brugia malayi in rhesus monkeys and in jirds. However, the levels of larval and adult recovery in both of these species is relatively low when compared with previously reported results using cats. This low rate of recovery, together with the more general distribution pattern in monkeys and the apparently pronounced migration in jirds, may be of importance in the selection of an experimental host for the study of Malayan filariasis. Of the several different vertebrates in which patent infection with Brugia malayi has been demonstrated, the domestic cat has most frequently been used for experimental studies. Recently we have described techniques for infection and recovery of this parasite that should facilitate future experimental studies using the cat as a model. However, at times it would be advantageous to have other models to use instead of, or in conjunction with, cats. It is commonly accepted that results from experiments using primates can more readily be extrapolated to man. Thus it was felt that if the techniques found to be useful in cats were found to be equally successful in a primate, certain studies such as preclinical drug studies or pathological studies could be carried out more effectively. Orihel and Pacheco (1966) demonstrated that at least the Philippine strain of B. malayi persisted in the rhesus monkey and therefore it might serve as a useful experimental host. On the other hand, a small animal Received for publication 20 May 1971. * This investigation was supported by the United States-Japan Cooperative Medical Science Program administered by the NIAID of the NIH, Department of Health, Education and Welfare. Grant No. AI 08260. t Permanent address: Box 32, Khartoum North, Sudan. model which would provide an inexpensive, easily handled host which could be used in drug screening programs would also be valuable. The jird, Meriones unguiculatus, has been shown to be a suitable host for Brugia malayi by Ash and Riley (1970). The present paper describes the results of distribution studies in rhesus monkeys and in jirds using methods previously found to facilitate observation and recovery of developing and mature Brugia malayi in cats. MATERIALS AND METHODS

Viability of a Self-Fertilizing Strain of Philophthalmus megalurus (Trematoda: Digenea)

A self-fertilizing strain of Philophthalmus megalurus was carried through 3 successive life cycles with little or no deleterious effects when compared with cross-fertilizing strains. No significant differences in the parameters of growth, eggshell formation, and the production of viable miracidia and cercaria were noted. However, significant reductions in recovery rates of adults from monometacercarial infections in the 2nd and 3rd generations were found. The effects of self-fertilization on different species of digenetic trematodes and cestodes and possible evolutionary adaptations for survival of self-fertilizing strains are discussed. The question of whether the hermaphroditic digenetic trematodes reproduce by selfor cross-fertilization has interested parasitologists for many years. Stunkard (1957) asserted that parasitic flatworms do not occur in interbreeding populations and reproduce by self-fertilization. Smyth (1962) suggested that self-fertilization is the most common type of reproductive process in the digenetic trematodes. On the other hand, Martin (1969) stressed the importance of intraspecific cross-fertilization for interpreting the biological species concept based on data from a study with Philophthalmus megalurus by Nollen (1968) and other studies utilizing different species of digenetic trematodes. The importance of cross-fertilization for development of the life cycle is another question to be answered now that it is known cross-insemination takes place in some species of digenetic trematodes. In the genus Philophthalmus the situation seems to vary among different species. Nollen (1971a) found that isolated adults of P. megalurus grow as well as those in multiple infections, and he was able to carry this eye fluke through one complete life cycle with no evidence of deterioration. On the other hand, Fried (1962) found that isolated adults of P. hegeneri stopped growing after 20 days in a chick's eye and never produced fertile eggs. Working with a similar Received for publication 20 May 1971. * This investigation was supported by the United States-Japan Cooperative Medical Sciences Program administered by the NIAID; Grant AI 09330. fluke, Howell and Bearup (1967) noted that single-worm infections of Philophthalmus burrili were infertile and contained no recognizable seminal receptacle. A similar situation in which species of a genus differ as to the viability of isolated adults also seems to occur in the genus Paragonimus. Sogandares-Bemal (1966) found that monometacercarial infections of cats with Paragonimus kellicotti never developed beyond the wandering preadult stage. However, Fan and Chiang (1970) found single infections of Paragonimus westermani in kittens and puppies developed into mature adults producing eggs capable of hatching. Varied results have been obtained with isolated adults of other digenetic trematodes. Bacha (1966) found that eggs produced by a second generation of isolated adults of Zygocotyle lunata in rats hatched less readily than those from multiple infections. Sogandares-Bernal (1965) found a higher percentage of paired adults of Microphallus opacus, which were excysted and maintained in vitro, produced eggs than those maintained in isolation. Viability of these eggs was not determined. Beaver (1937) and Lie (1965) observed normal miracidial development and hatching in single infections with species of Echinostoma. In this study successive life cycles of P. megalurus were investigated because this species grows and develops normally in isolated situations, and has proved viable for at least one self-fertilizing generation. A major drawback was the difficulty of raising the snail intermediate host, Pleurocera acuta. This was partially

Distribution of Developing and Mature Brugia malayi in Cats at Various Times after a Single Inoculation

Young cats were infected with Brugia malayi by administering 50 to 105 infective larvae to puncture wounds on the volar surface of one rear foot. At various times from 3 days to 69 weeks after exposure to the infective larvae, the animals were killed and the number and location of developing and mature worms recorded. Soon after infection a large proportion of the larvae could be recovered in the popliteal node. As the infection progressed, a substantial number of larvae left the popliteal lymph node and migrated distally in the lymphatic vessels toward the foot where infection had been initiated. In no instance was any appreciable migration to other body sites, including other lymph nodes, seen. In cats killed 7 weeks or longer after infection, 36 to 84% of the infecting dose was recovered at necropsy. The significance of these findings as they pertain to drug efficacy studies is discussed. In earlier studies, attempts were made to develop techniques which would facilitate rapid and consistent recovery of Brugia malayi from experimentally infected animals. It was found that if cats were infected on the rear foot by using a puncture wound technique, a relatively large proportion of the larvae which were administered could be readily recovered soon after infection. From 1 to 10 days after infection, larvae were almost exclusively located in the popliteal lymph node and adjacent afferent lymphatic vessels (Ewert and ElBihari, 1971). To utilize these techniques efficiently, it is necessary to determine if the parasites remain in the area where they are first recovered, or if they migrate to other sites as the larvae develop and reach sexual maturity. This would be of particular importance in studies correlating pathology with the site of infection and in determining the effect of drugs on the parasite. MATERIALS AND METHODS Materials and methods were similar to those described earlier (Ewert and ElBihari, 1971). Aedes togoi were fed on cats with from 40 to 300 microfilariae of subperiodic Brugia malayi per 20 mm3 of blood. Two weeks after feeding, the mosquitoes were dissected in a drop of physiological saline to free the infective-stage larvae. Eightto 14-weekold cats, reared in mosquito-free quarters, were anesthetized by intraperitoneal injection of 25 to 30 mg/kg sodium pentobarbital. Hair on the volar Received for publication 23 February 1971. * This investigation was supported by the United States-Japan Cooperative Medical Science Program administered by the NIAID of the NIH, Department of Health, Education and Welfare. Grant No. AI 08260. surface of 1 rear foot was cut and 100 puncture wounds were made in the skin with a small needle. A 0.03-ml drop of physiological saline was placed over these puncture wounds. Infective Brugia malayi larvae, freed from dissected mosquitoes, were immediately transferred individually to the drop of saline over the puncture wounds by using a small needle mounted on an applicator stick. Animals were not moved until the drop of saline had completely disappeared, approximately 30 min. At various times after exposure to infective larvae, cats were killed with an overdose of sodium pentobarbital. To demonstrate lymph nodes and vessels, 0.1 to 0.5 ml of a 0.4% aqueous solution of direct Sky Blue dye (Wyeth Laboratories) was injected into the foot pads, side of head, and base of tail, 15 to 60 min prior to killing the animals. Lymph nodes and superficial lymphatic vessels were exposed by reflection of the skin in the appropriate areas. After examining exposed nodes and lymphatic vessels with a stereoscopic :microscope, the nodes and as much of the vessels as possible were removed intact and placed in saline. Popliteal, inguinal, axillary, and cervical nodes along with their afferent and efferent vessels were teased with dissecting needles and examined for parasites. The intact living and dead parasites were removed and counted. A squash preparation between glass slides was then made of the nodes and lymphatic vessels and examined at higher magnification to detect any parasites missed earlier. Additionally, the rear legs and skin of rear quarters of all animals and the entire carcass and skin of several selected animals were soaked in warm saline for several hours. The saline was then decanted and the sediment examined for parasites.

Preferential Susceptibility of Male Jirds (Meriones unguiculatus) to Infection with Brugia pahangi

In two trials, 17 litter-mate pairs of jirds (Meriones unguiculatus) were inoculated subcutaneously in the groin with infective-stage larvae of Brugia pahangi to determine if sex of the host was a factor in establishing infection. The mean prepatent period for 14 male jirds ranged from 67 to 76 days. Only 2 female jirds developed peripheral microfilaremia, one at 87 and the other at 94 days postinoculation. In 3 other females, microfilariae were found only in cardiac blood. More adult worms were recovered from male animals than from females; for males the percentage of inoculum recovered was 1 to 32%, in females it was 0 to 18%. In males, adult worms were found primarily in the testes and the heart and pulmonary arteries, whereas adult worms occurred almost exclusively in the heart and pulmonary arteries of female jirds. It appears that male animals, for unexplained reasons, are more susceptible to infection with lymphatic-dwelling filarial worms than are females. Earlier studies on the development of Brugia pahangi and subperiodic B. malayi in jirds indicated that male hosts appeared to be more readily infected than females (Ash and Riley, 1970a, b). This report summarizes observations made on two series of litter-mate pairs of jirds inoculated with B. pahangi to determine if one sex was more susceptible to infection than the other. MATERIALS AND METHODS The origin of the strain of B. pahangi used here was as reported earlier (Ash and Riley, 1970a). Armigeres subalbatus and Aedes aegypti (Liverpool strain) served as vector mosquitoes and were fed on an anesthetized cat that had a microfilarial density of 100 to 200 per 20 mm3 of blood. Maintenance of mosquitoes and the recovery of infective-stage larvae from them were as previously described (Ash and Riley, 1970a). Litter-mate pairs of Meriones unguiculatus, 4 to 6 weeks old, from a colony maintained in the UCLA Vivarium, were used. Male and female animals were inoculated alternately by pipetting a known number of larvae into a subcutaneous pocket close to the left testis in males and into the left groin in females. Animals were bled at weekly intervals beginning 56 days after inoculation; the prepatent period was computed as the mean day between the last bleeding in which no microfilariae were found and the day on which microfilariae were first found. The techniques of bleeding and Received for publication 12 January 1971. * This investigation was supported by the United States-Japan Cooperative Medical Science Program administered by the NIAID of the NIH, Department of Health, Education and Welfare, U. S. Public Health Service Research Grant AI-07770. necropsy of infected animals were as previously described (Ash and Riley, 1970a).

Characterization of cell types in the parenchyma of Schistosoma mansoni.

SUMMARYA study has been made of the parenchyma of adult male Schistosoma mansoni by electron microscopy. Four different cell types have been characterized: one corresponding to cell bodies of the integument, two to muscle cells and another one to nerve cells with a possible neurosecretory function. The cytoplasm of the integument cell bodies is characterized by rod-shaped and round membrane-bound cytoplasmic bodies with a dense core, measuring 1200 to 2000 Å, similar to those found in the integument. One type of muscle cell has a large, clear nucleus, characteristic cytoplasmic vacuoles measuring up to 1 μm with a finely granular content and numerous free ribosomes. The other type of muscle cell is much smaller, with clumped nuclear chromatin and scanty perinuclear cytoplasm. Both types of cells give rise to muscle fibres which are morphologically distinct from the perinuclear cytoplasm. The nerve cells contain electron-dense, membrane-bound vesicles-ranging in size from 1000 to 3000 Å, similar to those found in nerve axons.Appreciable extracellular matrix has only been found under the integument and surrounding the processes of the integument cell bodies. This observation and the fact that silver methenamine after periodic oxidation stains intensely both the extracellular substance and the rod-shaped and round cytoplasmic bodies of the integument cells, suggests that these integument cells may be involve the production of extracellular matrix.This investigation was supported by grant AI 07806 from the U.S.P.H.S.The technical assistance of Mrs Lucille Jeffries and the photographic assistance of Mr William Douglas are acknowledged. This investigation was supported by the United States–Japan Cooperative Medical Science Program administered by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health, Department of Health, Education and Welfare.

Cholera research: United States--Japan cooperative medical science program.

Cholera research: United States--Japan cooperative medical science program.

Cholera Research: United States-Japan Cooperative Medical Science Program

Cholera Research: United States-Japan Cooperative Medical Science Program

Cholera Research: United States-Japan Cooperative Medical Science Program

Cholera Research: United States-Japan Cooperative Medical Science Program