Showing papers in "Journal of Parasitology in 1964"

Helminth parasites of the raccoon, procyon lotor in the southeastern united states.

Abstract: During the past 4 years examination of 32.0 raccoons, Procyon lotor, from five southeastern states revealed 39 species of helminth parasites. These were identified and grouped as follows: TREMATODA: 24 species; CESTODA: 3 species; NEMATODA: 11 species; and ACANTHOCEPHALA: 1 species. Differences were noted between the helminthic fauna of hosts from coastal and inland habitats. Helminthological studies on 320 raccoons from five southeastern states during the past 4 years have revealed some new parasitological information that is presented herein along with a summary of pertinent literature. Some comparable studies are those of Chandler (1942a), Babero and Shepperson (1958), and Jordan and Hayes (1959). Chandler recovered eight helminth species from 13 raccoons in east Texas and Babero and Shepperson reported 16 species of helminths from six raccoons in central Georgia. It is interesting to note that Jordan and Hayes obtained only seven species of helminths from 100 raccoons from Ossabaw Island, Georgia. Four species of protozoa, 27 nematodes, 20 trematodes, 7 cestodes, 3 acanthocephalans, and 15 arthropods have been reported as parasites of the raccoon (Clark and Herman, 1959). The hosts were obtained alive when possible. Many specimens were obtained from trappers during the regular trapping seasons. All animals were examined by conventional methods as soon as possible after capture. Exposure of the domestic cat to spargana was effected by mixing the larvae with canned cat food. Starved cats were fed macerated Received for publication 3 October 1963. * Contribution from the Zoology Department, North Carolina Agricultural Experiment Station, Raleigh. Published with the approval of the Director of Research as Paper No. 1702 of the Journal Series. Supported by research grants AI03209-04 and AI05927-01 from the National Institutes of Health, U. S. Public Health Service. 60 lung tissue containing metacercariae of P. procyonis in the same manner. RESULTS AND DISCUSSION Thirty-nine species of helminths, 23 of which were trematodes, were recovered from 320 raccoons. Tapeworms were represented bv three species, one of which, Spirometra mansonoides, was present as an adult in the intestine and as a sparganum in subcutaneous tissue. Eleven species of nematodes and one acanthocephalan were recovered (Table I).

Vertical migration of trichostrongylid larvae on grasses.

Abstract: Experiments were conducted in the laboratory to investigate the ability of trichostrongylid larvae to migrate vertically on various grasses under controlled environments. Only a small proportion, 2 to 3%, of the larvae given opportunity to ascend the grass blades, under conditions considered favorable for migration, climbed. Of these, 59.2, 26.7, 9.9, 3.4, and 0.8% were found on the 1st-, 2nd-, 3rd-, 4th-, and above the 5th-inch height level from the soil, respectively. It was concluded that though infective trichostrongylid larvae can climb grasses, the greatest portion remain at the base of the vegetation. The larvae were comparatively more successful on the tall fescue than on the smooth brome. The external morphology of grasses apparently influences migration of the larvae. A film of moisture is obviously advantageous to migration. On an average 0.65% of the test larvae climbed wetted blades while only 0.04% climbed unwetted blades. Upward migration was favored by higher relative humidity. On the average 1.36% of the test larvae were successful at 95% relative humidity while only 0.06% were successful at 56% relative humidity. Lower temperature adversely affected upward migration. Only 0.13% of the test larvae climbed at 40 F while 2.54% did so at 79 to 80 F. A direct influence of light intensity was not clearly established since migration in light and darkness was essentially similar. Taylor (1938), Rogers (1940), Kauzal (1941), Dinaburg (1944), Crofton (1948), and Rees (1950) studied vertical migration of infective trichostrongylid larvae on herbage. Their techniques essentially involved placement of known numbers of infective larvae, contained in a known volume of water, on the soil at the base of the test herbage in experimental plots in the field or laboratory. Subsequent recovery of larvae on the test herbage was interpreted to indicate vertical migration of the larvae from the soil surface onto the herbage. However, except for Crofton (1948) and Rees (1950), most of these investigators did not establish the actual height on the herbage the larvae ascended. Furthermore, only in a few cases (Crofton, 1948; Rees, 1950; Rogers, 1940) was the ability of the larvae to migrate vertically on the herbage correlated with environmental factors. The present study was undertaken in order to obtain an extensive series of observations Received for publication 20 August 1963. * From the Department of Veterinary Science, Paper N.S. 400, with the approval of the Director, Wis. Agr. Exp. Sta., supported in part by the University Research Committee. on the actual height of vertical migration by trichostrongylid larvae as well as the portion of the population which does migrate, and to observe the influence of various environmental factors including moisture, temperature, relative humidity, light, type of grass, and the number of grass blades available upon movement of the larvae. MATERIALS AND METHODS The larvae for these studies were obtained from "seed" calves primarily infected with Haemonchus placei with minor populations of Cooperia onchophora and Trichostrongylus colubriformis. The larvae were cultured in sterilized sphagnum according to the method of Cauthen (1940) and recovered by using Baermann funnels. Differential counts of larvae were not made for individual tests, and the larvae, therefore, have been referred to as trichostrongylid. The larvae were 7 to 20 days old when used in the tests, but in a given trial all larvae were the same age. Grass blades were grown from seeds planted in a light silt soil or a muck soil in paper pots. The soils were sterilized to render them free of nematode life prior to the tests. The grass seedlings emerged in a tuft of blades and were used when 3 inches or more tall. Numbers of larvae used in a test were estimated by dilution counts. By restriction of volume of the suspension, a known quantity of larvae could be placed in position on the soil surface at the

Competitive exclusion of Dactylogyrus extensus by Dactylogyrus vastator (Trematoda, Monogenea) on the gills of reared carp.

Abstract: Carp (Cyprinus carpio Linn.) infested with D. vastator, Nybelin, 1924, are slightly or not at all infested with D. extensus (Mueller and Van Cleave, 1932). Carp infested with D. extensus were found to be free or nearly free from D. vastator. These observations were made on 796 carp in ponds and subjected to statistical analysis. Under laboratory conditions in carp infested with D. extensus and subsequently exposed to D. vastator the latter species replaces the former, i.e., the number of D. extensus on the gills falls to a minimum or to zero even when the original infection with D. extensus was heavy. Histological changes of gills infested with D. vastator result in the loss of D. extensus and, eventually, in heavy infestations of D. vastator itself. With subsequent healing, the gills again become a suitable habitat for D. extensus and D. vastator. Eventually a specific resistance to D. vastator eliminates this species from the dactylogyrid fauna of the gills, and the habitat remains suitable for D. extensus and other dactylogyrids. Antagonism between different species of parasites to which fish are naturally exposed has been discussed by Dogiel (1958) and Bauer (1959). In a review of the subject the above authors cited the following examples: an increase in infestation with Lernaea (Copepoda) is associated with diminished susceptibility to glochidia and vice versa (Wilson 1916). Infection with Eubothrium crassum (Cestoda) prevents infection with Triaenophorus nodulosus (Cestoda) in trout (Scheuring, 1923). There is also antagonism between Octobothrium melangi (Monogenea) and Clavella devastatrix (Copepoda) in Gadus merlangus (Leigh-Sharp, 1933) and between Phyllodistomum folicum (Digenea) and Myxidium lieberkiihni (Neosporidia) in the urinary bladder of Esox lucius (Petrushevski, 1940). Ivasik (1953) found that Dactylogyrus extensus and D. vastator were antagonists to Trichodina carassii (Ciliata), while the above two species of Dactylogyrus and D. anchoratus were found Received for publication 23 April 1963. * This paper is part of a doctoral thesis carried out in the Department of Parasitology, Hebrew University, Hadassah Medical School, Jerusalem, under the direction of Professor G. Witenberg. t Supported by a grant from the Fish Breeders Association, Israel. 94 together on the gills of carp and apparently showed no mutual antagonism. The above observations were made during field studies. Little experimental work has been done on interspecific competition between parasites. The few experimental demonstrations of competitive relationships include those of Adler (1954) between Leishmania infantum and Plasmodium berghei, von Brand and Tobie (1960) working with species and races of Trypanosoma, and Holmes (1961) with Moniliformis dubius (Acanthocephala), and Hymenolepis diminuta (Cestoda). MATERIAL AND METHODS My observations are based mainly on an examination of 796 carp fingerlings collected in fish ponds infested with Dactylogyrus vastator and D. extensus during the spring of 1961-1962. The total length of the fingerlings was from 20 to 50 mm. Supplementary observations were carried out on fingerlings which were transferred to the laboratory and maintained in aquaria of various sizes at two constant temperatures (21 and 28 C). The parasites were counted on gills removed quickly from the living fish and immediately pressed between two glass plates. The results were subjected to the chi-square test.

Occurrence of Echinorhynchus clavula (Dujardin, 1845) nec Hamann, 1892 (Acanthocephala) in the fish of Llyn Tegid (Bala Lake), Merionethshire

Abstract: The grayling Thymallus thymallus, pike Esox lucius, roach Rutilus rutilus, and eel Anguilla anguilla of Llyn Tegid were examined for the occurrence of Echinorhynchus clavula; the total infection of these species of fish was 46.0%, 11.5%, 16.1%, and 27.7%, respectively. The degree of infection may be correlated with the feeding of the fish on Asellus meridianus, the only known intermediate host of E. clavula in Llyn Tegid, although in the case of the pike of the size range examined, a secondary establishment of the worms is postulated. The percentage occurrence of male E. clavula was lower than that of female worms. The production of shelled acanthors by female worms may be correlated with the degree of concentration of E. clavula in the fish intestine; thus, in the eel, where maximum concentration was found, maximum production of shelled acanthors was also found. No seasonal periodicity of occurrence and maturation of E. clavula was observed, shelled acanthors were seen during all months except February and December, and the failure to find worms with shelled acanthors in these months is believed to be a sampling deficiency. The literature relevant to the distribution of E. clavula and to the seasonal periodicity of occurrence and development of Acanthocephala is reviewed. It is postulated that temperature may play a significant role in the seasonal periodicity of some Acanthocephala of fishes. Llyn Tegid is a mesotrophic lake, 6 km in length by 1 km in width, situated about 45 km inland from Barmouth on the west coast of Wales. It is the largest Welsh lake and contains 13 species of fish (Chubb, 1963). This paper gives an account of the occurrence of Echinorhynchus clavula (Dujardin, 1845) nec Hamann, 1892 in grayling Thymallus thymallus (L.), pike Esox lucius L., roach Rutilus rutilus (L.), and eels Anguilla anguilla (L.). MATERIALS AND METHODS The fish (Table I) were captured by gill net, seine, or electric fishing. As soon as possible the alimentary tract of the fish was removed, slit longitudinally, and carefully searched. Acanthocephala found were placed in cold water until the proboscis was fully expanded. Fixation was by an alcoholformol-acetic medium as recommended by Van Cleave (1953). Various techniques were tried in order to obtain whole, stained, and cleared Acanthocephala. It was soon discovered that the use of an alcohol series for dehydration was unsatisfactory. Subsequently glacial acetic acid was used for dehydration (Chubb, 1962) and creosote for clearing. At first stains were used, but later were Received for publication 10 May 1963. 52 found to be unnecessary. The procedure finally adopted was as follows: fixation in AFA; dehydration via glacial acetic acid; clearing and preservation in creosote. Mounting in Canada balsam was unsatisfactory, as the fine detail of the unstained worms became invisible owing to excessive clearing. All specimens of E. clavula found were examined. The unstained material was found to be best for assessment of sex and stage of development of the female worms. The females were divided into two groups: one included those worms in which the phases of development of ovarian balls were present, but in which no shelled acanthors had been formed, while the other included worms in which shelled acanthors were observed.

The life history of neoechinorhynchus rutili and its development in the intermediate host (acanthocephala: neoechinorhynchidae).

Abstract: The life history of Neoechinorhynchus rutili (Mueller, 1780) was demonstrated experimentally. Shelled embryos are released by the adult worm into the lumen of the digestive tract of freshwater fishes and subsequently pass out with the feces. These "eggs" are ingested by ostracods (Cypria turneri). Within the intestine of the ostracod the acanthor hatches, penetrates the wall of the intestine, and in 6 to 12 days is found free in the hemocoel where it metamorphoses through the acanthella to the juvenile stage in 48 to 57 days. This stage is infective to the fish. The development in the ostracod has been followed and comparisons have been made with the other two species of the genus which have been studied. Neoechinorhynchus rutili (Mueller, 1780), an acanthocephalan worm parasitizing the small intestine of freshwater fishes, was redescribed by Van Cleave (1950). He showed its distribution to be continuous throughout the northern Holarctic Region, including Sweden, Finland, Russia, central Europe, Wisconsin, within the Arctic Circle of the Canadian Northwest Territories, off the coast of Alaska, and into Washington. The adult worm is found in 14 families of fishes: Salmonidae, Thymallidae, Cyprinidae, Catastomidae, Cobitidae, Esocidae, Gadidae, Gasterosteidae, Centrarchidae, Percidae, Labridae, Gobiidae, Cottidae, and Zorarcidae (Golvan, 1959). Van Cleave implied that occurrence of adults in frogs and turtles is probably due to accidental infections (1950). Life history studies on N. rutili have been sparse and incomplete. Robin (1870) found a worm in the leech Nephelis octoculata which Received for publication 23 August 1963. * This investigation was supported in part by research grant AI-00867 from the National Institute of Allergy and Infectious Diseases, U. S. Public Health Service and by the Oregon State Game Commission. Published with the approval of the Oregon State University Monographs Committee. Research Paper No. 460. t Present address: Department of Zoology, University of Wisconsin, Madison. was identified from his woodcut by Villot (1885) as Echinorhynchus clavaeceps (= N. rutili). In 1885, Villot reported finding E. clavaeceps encysted in the adipose tissue of the larvae of Sialis niger. Finally, in a research note Levander (1905) mentioned finding larvae in ostracods, but he neglected to record the genus and species. The early embryology of Neoechinorhynchus rutili has been studied in detail by Meyer (1931). He traced the cleavage and cell lineage up to the development of the shelled acanthor, which is shed by the adult female and is infective to the intermediate host. No study has been made of the complete development in the intermediate host. There have been, however, a few developmental studies carried out on other species of Acanthocephala. The scattered observations made prior to 1940 have been discussed by Ward (1940). Hynes and Nicholas (1957) brought together subsequent observations in their work on Polymorphus minutus. Moore (1962), in his study of Mediorhynchus grandis, pointed out the need for more information about the acanthor types and their development to aid in establishing phylogenetic relationships. The present study was undertaken to determine experimentally the life cycle in local hosts and to describe the development in the

The Protective Action of Chaetogaster limnaei on Snails exposed to Schistosoma mansoni.

Abstract: Experiments demonstrated that infestation with the oligochaete worm, Chaetogaster limnaei, afforded a degree of protection to the snail Australorbis glabratus when the snail was exposed to Schistosoma mansoni miracidia. To a lesser extent, the oligochaetes protected snails exposed to echinostome cercariae. The presence of C. limnaei within the kidney of the snail Physa heterostropha is reported. An intimate association between the oligochaete worm Chaetogaster limnaei von Baer and various species of aquatic snails has long been recognized (von Baer, 1827), yet the nature of this relationship has not been adequately defined. Early naturalists considered C. limnaei to be a true parasite which was thought to feed upon the "slime" produced by the host. However, Wagin (1931) and subsequent investigators demonstrated that the oligochaete fed principally on microorganisms, and thus the worm has been considered a commensal. Wagin also observed that the oligochaete ingested cercariae and suggested that C. limnaei might be of value in controlling trematode transmission. While Krasnodebski (1936) confirmed Wagin's field observations and experimentally demonstrated that the oligochaete would ingest various types of cercariae, he did not believe that Chaetogaster could play a significant role in the control of trematodes. Backlund (1949) observed that cercariae of Fasciola hepatica were ingested by Chaetogaster, and Ruiz (1951) later reported a similar observation with respect to Schistosoma mansoni. Coelho (1957) noted that Chaetogaster frequently ingested S. mansoni miracidia, but commented that the worms did not protect snails from infection. HowReceived for publication 3 March 1964. * These studies were supported in part by Grants AI-00513 and 5 TI AI 46 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Public Health Service, Bethesda, Maryland. 441 ever, Khalil (1961) presented circumstantial evidence suggesting that snails infested with C. limnaei were refractory to infection with Fasciola hepatica under laboratory conditions. The present study was initiated to determine quantitatively whether Australorbis glabratus infested with C. limnaei were protected against invasion by miracidia of S. mansoni and by cercariae of an echinostome. MATERIALS AND METHODS Specimens of C. limnaei were obtained from Physa heterostropha collected in Boston. The oligochaetes were freed by immersing the snails in 1% Urethan (ethyl carbamate) for 3 to 5 min. Anesthetized worms were collected by pipette, allowed to recover in charcoal-filtered tap water, and used immediately or colonized for later use (Brandwein, 1937). A Puerto Rican strain (PR-1) of Australorbis glabratus was used in all experiments. This snail strain has been maintained in our laboratory for several years and has been free from infestation with C. limnaei. Miracidia were hatched from eggs obtained from the livers of mice infected with a Puerto Rican strain of S. mansoni and were used within 30 min of emergence (Michelson, 1964). Cercariae of an unidentified echinostome were obtained from Physa heterostropha collected from local ponds. Five C. limnaei were added to individually isolated A. glabratus in 5-ml beakers containing 3 ml of charcoal-filtered tap water. The snail was confined with the oligochaetes for 1 hr or until all worms became attached. At this time and depending upon the experiment, one to five miracidia or ten cercariae were added to each beaker. After an exposure of approximately 16 hr, the snails from any one experiment were maintained together in a battery jar containing 2.5 liters of water. The battery jars were continually aerated, maintained at 25 ?+ 1 C, and the snails fed Romaine lettuce. ConThis content downloaded from 157.55.39.157 on Tue, 17 May 2016 04:47:34 UTC All use subject to http://about.jstor.org/terms 442 THE JOURNAL OF PARASITOLOGY, VOL. 50, NO. 3, JUNE 1964 TABLE I. Influence of Chaetogaster limnaei infestation on the rate of infection of snails exposed to S. mansoni miracidia and to echinostome cercariae. Snails with C. limnaeil Control snails Group no. No. positive/ No. positive/ no. exposed no. exposed 5 miracidia of S. mansoni per snail 12 2/7 4/8 22 7/18 14/20 32 1/5 5/6 4 5/10 8/10 5 2/10 10/10 Total 17/50 (34%) 41/54 (76%) 1 miracidium of S. mansoni per snail 6 1/10 5/10 7 2/15 7/15 Total 3/25 (12%) 12/25 (48%) 10 cercariae of echinostome sp. per snail 8 7/10 10/10 9 7/10 10/10 Total 14/20 (70%) 20/20 (100%) 1 All snails infested with 5 worms each. 2 Test groups 1, 2, and 3 had one, two, and one snails die before they could be examined for the presence of infection; these have not been included with the data. trol snails, free of C. limnaei, were exposed to miracidia or cercariae in the same manner. Two weeks postexposure, control and Chaetogaster-infested snails were examined for the presence of S. mansoni sporocysts by the technique of Chernin and Dunavan (1962). Snails exposed to echinostome cercariae were examined 7 days postexposure for the presence of metacercariae. For this purpose the snail was removed from its shell, the mantle cut along its left margin and reflected to the right, and the pallial cavity examined under a 13 X dissecting microscope. Experimental results In five experiments a total of 54 snails, each infested with five Chaetogaster limnaei, were exposed individually to five S. mansoni miracidia and 17 (34%) of 50 snails which survived were found infected (Table I). By comparison, in the control groups of snails 41 of 54 snails (76%) were infected. Thus, a mean reduction in the rate of infection of 42% was associated with the presence of C. limnaei. In two additional experiments (Table I), snails harboring five C. limnaei were exposed individually to a single miracidium of S. mansoni. Of a total of 25 snails so exposed, three (12%) became infected. An infection rate of 48% (12/25 snails) occurred, however, in the two groups of control snails. A mean reduction of 36% in the infection rate was again observed to be associated with the presence of C. limnaei. Snails were exposed to echinostome cercariae in two experiments (Table I), and all the control snails were later found infected. However, only 70% (14/20) of the snails infested with C. limnaei had metacercarial cysts. In the first experiment 38 and 58 metacercarial cysts were recovered from the Chaetogaster-infested and control snails respectively, and in the second experiment 22 and 69 cysts respectively. Infected snails were found to have from three to eight cysts each. Miscellaneous observations The intensity of Chaetogaster infestation in snails under field conditions would appear to vary considerably. Backlund (1949) observed that no more than ten Chaetogaster were found on specimens of Lymnaea stagnalis and L. ovata; however, Krasnodebski (1936) noted as many as 300 worms on a single specimen of L. stagnalis, up to 60 worms on Physa fontinalis, and a mean of 1.3 worms on specimens of Ancylus lacustris. In our own experience, as many as 25 C. limnaei have been recovered from a single Physa heterostropha. In one collection of 105 P. heterostropha there was a mean of nine worms per snail. The heaviest infestations were found on older and larger snails, particularly those which had overwintered. Quantitative data concerning natural infestations of Chaetogaster on the snail Auistralorbis glabratus are not available, although infestations of field populations have been reported (Coelho, 1957). Natural infestations of South African populations of Bulinus tropicus, Bulinus africanus, and Biomphalaria pfeifferi have also been noted by Bayer (1955). Likewise, laboratory colonies of A. glabratus, as well as Bulinus truncatus and B. tropicus, have been observed to be infested with Chaetogaster (Khalil, 1961). Although it has generally been thought that C. limnaei acts as a commensal of snails, feeding principally on aquatic microorganisms, there is some evidence to suggest that this oligochaete may also be a parasite. The observation of Lankester (1870) of C. limnaei in the kidney of a single L. stagnalis has been overlooked. Our examination of both field and laboratory populations of P. heterostropha This content downloaded from 157.55.39.157 on Tue, 17 May 2016 04:47:34 UTC All use subject to http://about.jstor.org/terms MICHELSON-PROTECTIVE ACTION OF CHAETOGASTER ON SNAILS EXPOSED TO SCHISTOSOMA MANSONI TABLE II. Distribution of Chaetogaster limnaei in a collection of Physa heterostropha. Number C. limnaei present Snail no.1 On body surface and Inside in mantle cavity kidney 1 1 0 2 0 7 3 8 4 0 5 5 2 1 6 6 5 7 1 3 8 0 7 9 5 10 1 1 2 5 12 0 2 3 3 1 14 1 5 5 5 18 1 Five snails in this collection had no Chaetogaster and are not included in the table. revealed that under some circumstances large numbers of C. limnaei may be found within the kidney of the snail. In one group of P. heterostropha, examined 3 days after collection, 15 of 20 snails were infested. Fourteen of the 15 infested snails harbored 1 to 18 worms within the kidney; whereas only nine of these 15 snails had external infestations (Table II). Of further interest is the fact that snails could be free of external infestation while harboring numerous worms in their kidney. Histologic studies demonstrated that most of the oligochaetes were in the anterior portion of the kidney, but any part of the organ might harbor the worms (Fig. 1). No tissue reactions were observed in sections of snails so infected, although some oligochaetes appeared attached to the renal epithelium and renal concretions were observed in the intestinal tract of a few worms. Oligochaetes were not found in the kidney of experimentally infested A. glabratus.

Effect of x-irradiation upon the infective larvae of ancylostoma caninum and the immunogenic effect in dogs of a single infection with 40 kr-irradiated larvae

Abstract: X-irradiation of the infective larvae of Ancylostoma caninum reduces the infectivity of the larvae as measured by subsequent intestinal establishment of adult hookworms. As the dose of radiation is increased the infectivity of the larvae is decreased and the pathogenicity to the host of the resulting burden of hookworms is reduced. Male larvae are more sensitive to the effects of X-irradiation than are female larvae, particularly at the higher levels of irradiation. At radiation doses of 40 kr and greater the female worms in the resulting population are invariably sterile. A single subcutaneous vaccination of dogs aged 3 months with 1,000 40 kr-irradiated larvae confers a highly significant resistance. This vaccination confers highly satisfactory resistance to the pathogenic effects of such a challenge, when measured in terms of hematological, clinical, and coprological changes. In a number of host-helminth systems it has been shown that the previous administration of X-irradiated infective larvae will protect the host against a potentially pathogenic challenge of normal larvae. The present state of these helminth vaccines has been surveyed by Urquhart et al. (1962). The work of Herrick (1928), McCoy (1931), Foster (1935), Otto and Kerr (1939), and Otto (1941) has demonstrated that dogs can be immunized against subsequent severe challenge of Ancylostoma caninum by the administration of normal infective larvae. This resistance to challenge was manifested by reduced worm establishment from the challenge of normal larvae, and in some cases also by a reduction in worm egg output. Cort and Otto (1940) and Otto (1948) have surveyed the literature on the subject of experimental vaccination against ancylostomiasis in dogs. Many of the recorded results describe experiments in which stray or "pound" dogs of unknown age and originating from hookworm enzootic areas were used. Resistance shown to challenge infection in these often uncontrolled experiments, ascribed by the author to Received for publication 27 April 1964. be a result of either age or acquired immunity, appears retrospectively and in the light of current knowledge to have been, in many cases, a combination of both manifestations of resistance. This paper describes the effects of X-irradiation on the parasite and an attempt to immunize dogs by a single vaccination regime using such irradiated larvae. MATERIALS AND METHODS Since this is the first of such reports, the details of technique are described in full. Acquisition and preparation of experimental animals Dogs of mixed breeding were purchased from dealers as newly weaned litters at the age of 6 weeks and were isolated in small groups until approximately 3 months of age. During this period they were vaccinated against canine distemper, contagious canine hepatitis, and leptospiroses canicola and icterohaemorrhagiae (Canilep-Glaxo Laboratories, Ltd., Greenford, England) and were treated twice with piperazine adipate for ascarids (Coopane-Cooper, MacDougall and Robertson, Ltd., Berkhamsted, England). Repeated fecal examination of all dogs after purchase and prior to any anthelminthic medication has demonstrated the absence of strongyle eggs and hence precludes the possibility of prior exposure to hookworm (including Uncinaria stenocephal7).

Experimental Toxocara canis Infections in Chickens and Pigeons.

Abstract: Studies were made to determine whether chickens and pigeons could serve as paratenic hosts of Toxocara canis. It was found that both types of hosts harbored living infective-stage larvae for at least 142 days after inoculation. At this time, of the number of larvae inoculated, it was possible to recover approximately 30% from chickens and 18% from pigeons. Human infections with larvae of Toxocara canis are known to occur throughout the United States and in Mexico, Europe, Australia, the Philippines, and South Africa (Beaver, 1962). In the majority of cases, the infected individual is a 1to 3-year-old child who acquired the infection by ingesting infective eggs in contaminated soil (Beaver et al., 1952). In some cases, however, especially those in which the eye is invaded, the infected individual is an older child or young adult (Beaver, 1962). Although in such instances the invading larvae possibly have persisted elsewhere in the body since early childhood, it is also possible that they were acquired by eating raw or poorly cooked parts of infected meat animals. T. canis larvae persist for months or years in a wide range of mammalian laboratory hosts (Beaver, 1962), and it is presumed that they are transferred from animal to animal through paratenic hosts in nature (Sprent, 1954). It has been suspected that the eosinophilia observed in persons eating raw liver for the control of pernicious anemia was caused by infection with nematode larvae, possibly Toxocara, harbored in the liver (Beaver, 1956). Done et al. (1960) found that in the experimentally infected pig, many infective-stage Received for publication 21 August 1963. * Supported by grants EF-, EPD-14573, and AI04919 from the National Institutes of Health, U. S. Public Health Service. t Present address: Department of Veterinary Parasitology, Agricultural and Mechanical College of Texas, College Station. larvae of T. canis could be recovered from the liver, brain, lungs, and muscles more than 2 months after inoculation. In a similar study on T. canis in sheep and lambs, Schaeffler (1960) recovered the larvae from liver, muscle, and other tissues 3 months after inoculation. Studies on poultry have been brief and inconclusive. Sprent (1956) recovered six larvae of T. cati from the carcass of an experimentally infected chick at 10 days but none could be found in others at 15, 20, or 25 days. Beaver (1956) found that larvae of T. canis persisted for at least 3 months in the liver of chickens and pigeons. The present study was undertaken to get further information on the persistence and distribution of T. canis larvae in the tissues of chickens and pigeons. MATERIALS AND METHODS T. canis eggs obtained by sedimentation of feces from naturally infected dogs were incubated in 1% formalin at room temperature for 4 to 6 weeks before use. Estimated numbers of infective eggs were injected by pipette directly into the crop of young chickens and pigeons. Recovery of larvae was accomplished by two procedures: by one, the tissues were ground in saline with a Waring blendor and digested in 1% pepsin (pH 1.5) in saline (0.83%) at 37 C for 3 to 4 hr; suitable aliquots were then washed and concentrated by centrifugation in saline and the sediment was examined microscopically for larvae. By the other, tissues were chopped and finely minced with scissors and scalpel, placed in a Baermann apparatus in which the suspending medium was pepsin digest fluid, allowed to stand at 37 C for 3 to 4 hr, after which the sediment was collected and examined in the usual manner. This method, though

Structure of trichomonas vaginalis donn'e.

Abstract: The structure of Trichomonas vaginalis is described from direct smears of vaginal secretions and from axenic cultures. The employment of protargol stain and observations of living organisms with the aid of phase contrast clarified some of the hitherto inadequately understood morphological details and relationships. The former method was particularly helpful in revealing the structure of the parabasal apparatus and of the axostylar capitulum. The latter allowed a study of the motion and of the formation of pseudopodia; it was also found superior for the analysis of the distribution of cytoplasmic inclusions such as the paraxostylar and paracostal granules. The discussion includes a comparison of the urogenital trichomonad of man with the two closely related species, Trichomonas tenax and T. gallinae. Trichomonas vaginalis Donne is a protozoan parasite commonly found in the urogenital tract of men and women. As far as is known this flagellate is limited in its distribution to human hosts. It may be stated without fear of exaggeration that only a few parasitic protozoa have been the subject of more extensive morphological and physiological study than the trichomonad in question. Yet there seems to be no account of the structure of this species based on observations of living material with the aid of phase contrast or of preparations stained with protargol. Among the techniques employed previously in the studies of T. vaginalis the iron-hematoxylin method of staining gave the most satisfactory results. Through the use of this technique many workers were able to describe accurately numerous morphological details of the flagellate. Of the more recent investigators, Wenrich (1931, 1939, 1944, 1947), Powell (1936), and Hawes (1947) contributed most significantly to the understanding of the structure of T. vaginalis. Despite the great interest in this trichomonad there appear to be only two apparently preliminary reports (Inoki et al., 1959, 1960) of its fine structure, Received for publication 1 April 1964. * This investigation was supported by Research Grant AI-00742 from the National Institute of Allergy and Infectious Diseases, Public Health Service. and there can be no question of the pressing need for further electron microscopic studies of this and all the other trichomonad species. The present report is designed to supplement and complement the previous morphological accounts of T. vaginalis by describing numerous strains of this species on the basis of observations of living material with the aid of phase contrast and of fixed preparations stained not only with iron hematoxylin (or hematein), but also with protargol. Also, some mensural considerations have been included which may aid further in the establishment of the morphological identity of the urogenital trichomonad

Life cycle and development of prosthorhynchus formosus (van cleave, 1918) travassos, 1926, an acanthocephalan parasite of birds.

Abstract: The life cycle of Prosthorhynchus formosus (Van Cleave, 1918) Travassos, 1926 is presented. The terrestrial isopods Armadillidium vulgare, Porcellio laevis, and P. scaber served as experimental intermediate hosts, and chickens and turkeys as experimental definitive hosts. After ingestion of the eggs by the isopod, the acanthor emerges from its shells within 15 min to 2 hr, enters the gut wall of its host, and remains there 15 to 25 days. It then migrates to the hemocoel and develops through the acanthella stage to the infective cystacanth by the 60th to 65th day of infection. Van Cleave (1918) described Plagiorhynchus formosus on the basis of four specimens collected from the flicker, Colaptes auratus, at Bowie, Maryland. In 1926, Travassos reassigned this species to the genus Prosthorhynchus but gave no reasons for the change. Van Cleave (1942) disagreed strongly with this change in generic status and reaffirmed its position in Plagiorhynchus. Golvan (1956) clarified the differences between Plagiorhynchus and Prosthorhynchus and defended its position in Prosthorhynchus. This view, which was upheld by Petrochenko (1956) and Yamaguti (1963), is accepted by the present authors. Jones (1928) extended the host list to include the chicken (Gallus domesticus), robin (Turdus migratorius), and the crow (Corvus americanus). She remarked on the possibilities of this worm becoming an important parasite of domestic fowl. Cuvillier (1934) added the catbird (Dumatella carolinensis), and a thrush Received for publication 17 April 1964. * Part of a thesis submitted by the senior author to the Graduate School of Colorado State University in partial fulfillment of the requirements for the Ph.D. degree, February 1964. This investigation was supported by Training Grant PHS-2E-94 (C2,3) from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health, U. S. Public Health Service, and by a fellowship from the Boettcher Foundation, Denver, Colorado. (Hylocichla sp.), and Van Cleave (1942) the towhee (Pipilo erythrophthalmus), starling (Sturnus vulgaris), grackle (Quiscalus quiscala), and hermit thrush (Hylocichla guttata). Chandler and Rausch (1949) found it in the brown thrasher (Toxostoma rufum), and Hunter and Quay (1953) in Macgillivray's seaside sparrow (Ammospiza maritima macgillivaraii). The present report adds the red-shafted flicker (Colaptes cafer) and the domestic turkey (experimental). Sinitsin (1929) reported finding a juvenile of Plagiorhynchus formosus in the sowbug, Armadillidium vulgare, near Washington, D. C. To date, this is the only report on the life history of P. formosus, although Dollfus and Dalens (1960) reported larvae of Prosthorhynchus cylindraceus in A. vulgare in France. This work presents the complete life cycle of Prosthorhynchus formosus as observed under laboratory conditions, utilizing three species of terrestrial isopods as intermediate hosts and domestic fowl as definitive hosts. MATERIALS AND METHODS

Maintenance in vitro of Larval Schistosoma mansoni in Tissues from the Snail, Australorbis glabratus.

Abstract: Portions of digestive gland and ovotestis were removed from aquarium-reared Australorbis glabratus about 40 days after infection with Schistosoma mansoni. The tissues were minced and explanted in flasks under sterile conditions. The cultures were maintained at 26 to 28 C either on a complex medium, or, with generally superior results, on a balanced salt solution containing glucose and trehalose (BSS). Medium was changed daily and cultures were evaluated by counting the emerged cercariae and by histologic study of the tissues. Apparently normal cercariae emerged daily in relatively large numbers during the 1st week, and in decreasing numbers during the succeeding 1 to 2 weeks. Omission of the sugars from BSS adversely affected the yields of cercariae; addition of amino acid mixtures to BSS did not increase the amount or duration of cercarial emergence. At least in part, cercarial emergence was found to be influenced by light. A small proportion of culture-derived cercariae was infective for mice by skin penetration or intraperitoneal injection. Tissues harboring "immature" infections were also explanted, i.e., from snails 19 days after exposure to S. mansoni. It was demonstrated that cercarial embryos were capable of undergoing maturation in vitro when cercariae appeared in these cultures after a lag of 8 to 13 days. Intramolluscan larval trematodes have received little study in vitro. The few published accounts indicate that immature rediae of Cyclocoelum microstomum from miracidia survived at most for 14 days in complex media (Ingersoll, 1956), and that rediae of Fascioloides magna from snails survived up to 10 days in solutions of individual substrates (Friedl, 1961). The axenic rediae did not grow or produce succeeding larval stages in vitro. Techniques are now described for the study in vitro of larval Schistosoma mansoni in tissues from parasitized Australorbis glabratus. It has been demonstrated, apparently for the first time, that trematode cercariae can develop and emerge in vitro and that under certain circumstances some of them are infective. MATERIALS AND METHODS Maintenance and infection of snails Snails were from a Puerto Rican strain (PR-2) of A. glabratus colonized in our laboratory since 1957 (Chernin and Schork, 1959) and maintained on watercress and romaine lettuce. Groups of 100 snails, 4 to 8 mm in diameter, were mass-exposed in finger bowls to miracidia of a Puerto Rican strain of S. mansoni (Chernin and Dunavan, 1962). Snails were then maintained on lettuce at 25 C, in darkness, in covered, aerated, stainless-steel pans. The snails were isolated in vials and examined for cercarial emergence 38 to 41 days postexposure, except as noted. Snails yielding cercariae at this time were used in experiments within 24 hr; these usually measured 12 to 16 mm in diameter. Composition of solutions Two solutions, previously developed in snail tissue culture studies (Chemrnin, 1963), were compared as media. The first is a complex nutrient medium (TC-NM) consisting mainly of a balanced salt solution and sugars (BSS), plus small amounts of vertebrate serum, amniotic fluid, embryo extract, lactalbumen hydrolysate, and yeast extract. The second solution is the BSS which contains several salts and 100 mg each of glucose and trehalose per 100 ml. The TC-NM was adjusted before use to pH 7.4 to 7.5 with 0.1 N NaOH; the BSS required no adjustment for a similar pH. All media were supplemented with 200 units penicillin G and 200 pg streptomycin sulfate per ml. Securing and processing snail tissues Glassware and instruments were sterile, all procedures were done in a hood, and a stereomicroscope was used in dissection. Snails were blotted dry, wiped with 70% alcohol, and kept temporarily Received for publication 27 April 1964. * These studies were supported in part by research grant AI-00513 and by a Research Career Award from the National Institute of Allergy and Infectious Diseases, U. S. Public Health Service.

Incidence of trypanosoma cruzi (chagas) in wild vectors and reservoirs in east-central alabama.

Abstract: TrypanOsoma cruzi (Chagas) was isolated from 17 of 126 (13.5%) opossums and 5 of 35 (14.3%) raccoons from east-central Alabama. Inoculation of blood, heart muscle, kidney tissue, urine, and peritoneal fluid from survey animals into NNN or Chang's medium showed that the organism was most often isolated from heart muscle followed by blood, kidney, peritoneal fluid, and urine. The trypanosome was recovered from 11 of 181 (6%) triatomid bugs collected in the same area. Of the 44 collections, 32 were from hollow trees or stumps, 10 were from human habitations, and 2 from the quarters of domestic animals. In some instances, both infected vectors and reservoirs were found in heavily populated areas. Trypanosoma cruzi (Chagas), the etiologic agent of Chagas' disease, was first reported from triatomid bugs and man by Chagas (1909). Subsequent reports showed the organism to be present in many areas of Latin America. The first report of T. cruzi in the United States was that of Kofoid and McCulloch (1916), who found the flagellate in triatomid bugs in California. At the time, they named the organism Trypanosoma triatomae since they could not find the organism in the blood of wood rats upon which the bugs were feeding. Later reports verified the presence of T. cruzi in vertebrate reservoirs and triatomid bugs in the southwestern United States (Kofoid and Donat, 1933; Kofoid and Whitaker, 1936; Wood and Wood, 1941). Woody and Woody (1955) reported the first naturally acquired human case of Chagas' disease in the United States in a child from Corpus Christi, Texas. In the same year, a second indigenous case, also diagnosed in a child, was reported from Bryan, Texas, according to a report by Goble (1958). Walton et al. (1956) reported the isolation of T. cruzi from three raccoons captured in Maryland; however, they were unable to find triatomid vectors in the area where the raccoons were captured, according to a report in 1958. McKeever et al. (1958) reported finding an organism resembling T. cruzi in 107 animals of four species in southwestern Georgia and northwestern Florida. Later research verified that the organism was T. cruzi (Norman et al., 1959). Yaeger (1961) found inf cted triatomid bugs in areas of Louisiana where mammals harboring trypanosomes had been collected. These reports indicate that T. cruzi is probably present throughout the southeastern United States. This paper reports the incidence of T. cruzi in wild mammals and triatomid bugs collected in east-central Alabama between September 1960 and June 1963.

Geographic distribution of some species of trichodinids (Ciliata: Peritricha) parasitic on fishes

Abstract: North American Trichodina from Carassius auratus, Lepomis cyanellus, L. macrochirus, Micropterus salmoides, and Rhinichthys atratulus have been studied by the silver-impregnation method and compared with European species. T. reticulata Hirschmann and Partsch, 1955, and T. (Foliella) subtilis Lorn, 1959, originally described from European goldfish, are herein recorded from North America also. T. fultoni Davis, 1947, from Lepomis cyanellus, Micropterus salmoides, and Rhinichthys atratulus is redescribed, and T. domerguei f. magna Lorn, 1961, described from European tench and gudgeon, is considered a synonym. A Trichodina sp. from the gills of Lepomis macrochirus is described; it is almost identical with T. nigra Lom, 1961 from European Rutilus, T. discoidea from centrarchids, and T. tumefaciens from Cottus of North America. Other North American trichodinids are reviewed briefly. Up to the present time, more than 90 species of Trichodina have been described from the gills and skin of marine and freshwater fishes. Many of them have been considered as new only because they were found in a different host and remote geographic area. The descriptions in many cases were inadequate since the uniform body structure of these ciliates yields few characters for solid differentiation of species. However, the recently employed Klein's silver-impregnation technique (Lorn, 1958; Raabe, 1959) clearly reveals details of the adhesive disc which are important features of urceolariid taxonomy. This, together with careful biometrical and other morphological information, represents a good base for taxonomic studies of this group. It is beyond the scope of this communication to present all the problems of trichodinid taxonomy; they can be found in Uzmann and Stickney (1954), Raabe (1959), and Lorn (1958, 1961, 1962). Though insufficient description of some species does not permit us to identify them again, reexamination of a number of species which have been accurately characterized by older methods is possible. This is a study on the geographic distribution of some fish trichodinids, based on a comparison of four species Received for publication 9 April 1963. 30 from some North American fishes from Pennsylvania and West Virginia with material from Cz choslovakia. Trichodina reticulata Hirschmann and

In vitro cultivation of spirometra mansonoides (cestoda) from the procercoid to the early adult.

Abstract: Spirometra mansonoides has been grown in vitro from the procercoid to the young adult using the methods of Mueller (1959) and Berntzen (1961, 1962, 1964). The process involves scolex differentiation, shedding of the larval body, growth from 0.1 mm to 64 mm in length, and segmentation to form the strobila. Successful cultures had a gas phase of 95% N2/5% CO2 or 90% N2/10% CO2 and were preceded by an evaginating solution containing bile salts and trypsin. The medium is complex but does not contain chick embryo extract. Incubation temperature was 39 C. Oxygen is toxic to the cultures. Lack of C02 or omission of the evaginating solution resulted in no growth. Essentially twothirds of the life cycle of this tapeworm has now been accomplished in vitro in the absence of the normal hosts: snake or mouse, and cat. It has been demonstrated that the tapeworms, Hymenolepis diminuta and H. nana, can be grown from the larval to the ovigerous adult stage in vitro using culture apparatus and media described by Berntzen (1961, 1962, 1964). Mueller previously succeeded in culturing the plerocercoid larva of Spirometra mansonoides in vitro from the procercoid stage using methods described in a series of papers (Mueller, 1958, 1959a, 1959b, 1959c). In vitro-reared plerocercoids proved to be infective for cats, and adult worms resulting from them were normal in every detail. Furthermore, the culture system could be made to serve as a surrogate host to the plerocercoid Received for publication 28 May 1964. *Supported by Research Grants Numbers AI04175-01 and AI-01876-06 from the United States Public Health Service, National Institutes of Health, Bethesda, Maryland. t Department of Medical Microbiology and Immunology. t Department of Microbiology. over several successive generations with no indication of impairment to the worm. Since Hymenolepis is a cyclophyllidean genus, the question arose whether the culture methods developed by Berntzen would prove equally successful for rearing a pseudophyllidean cestode from the plerocercoid to the adult stage in vitro. Smyth (1946, 1949) succeeded in bringing about the differentiation to sexual maturity and oviposition of several larval pseudophyllideans (Ligula, Schistocephalus) in vitro, but these large plerocercoids, from the body cavity of fishes, have abundant amounts of stored energy, and little or no increase in mass is involved. In these larvae the primordia of the genitalia are already present, and in Schistocephalus even segmentation is complete. Smyth (1959) also succeeded in inducing differentiation of a small plerocercoid from fishes (Diphyllobothrium dendriticum ?) in vitro. In this case he obtained segmentation and development of genital primordia, but there was no increase in mass, and the resulting

Fine structure of the eyespot in the miracidium of philophthalmus megalurus (cort, 1914).

Abstract: Miracidia from worms reared in the eyes of chicks were prepared as sections for light and electron microscopy. The eyespot consists of two closely apposed pigment-cup cells, each occupied by two sensory cells with one anterior and the other posterior to a vertical septum formed by the cup cell. This orientation extends also to the rhabdomeres of the two sensory cells in each cup and facilitates interpretation of the phototactic behavior of the miracidium. The fine structure of photoreceptors has been described for a variety of animals, but evidently the use of electron microscopy to investigate such receptors in larval trematodes has been limited to two species of miracidia. Kiimmel (1960) described the eyespot in that larva of Fasciola hepatica, and studies dealing with the miracidium of Philophthalmus megalurus were reported in a brief abstract by the author (Isseroff, 1963). This paper presents more fully the observations included in that part of the abstract concerned with fine structure of the eyespot. In reporting the life history of this species in Indiana, West (1961) identified it as Philophthalmus gralli, which had previously been found in Indochina and Formosa. In Hawaii, Alicata (1962) also identified as P. gralli, a trematode whose life history differed in several respects from that reported by West. However, Cable and Hayes (1963) determined that West and Alicata were dealing with different species and designated the North American one as P. megalurus (Cort, 1914) because the specific name of its larva, Cercaria megalura, has priority over other names that may have been given to the same species.

Effect of certain laboratory procedures on virulence of the Jones' Barn strain of Trichomonas gallinae for pigeons.

Abstract: Continued in vitro cultivation at 37.5 C of the highly virulent Jones' Barn strain of Trichomonas gallinae was accompanied by gradual loss of pathogenicity for Trichomonas-free, nonimmune domestic pigeons. This virulence loss was about twice as rapid when the organisms were isolated with the use of penicillin and dihydrostreptomycin as when they were isolated without such agents. Pathogenicity was rapidly restored, however, to its original virulence level by serial pigeon-topigeon passage of the trichomonads. Whereas attenuation occurred in organisms maintained at incubator temperatures, virulent trichomonads frozen and maintained at subzero temperatures (-19 C and -72 C) showed no decrease in pathogenicity when thawed and placed in appropriate pigeons following 52 weeks at -72 C. Trichomonads maintained at 37.5 C for 98 weeks became so attenuated that they failed to elicit an immune response in infected birds, a reaction characteristic of normal, avirulent T. gallinae. Both Bos (1934) and Gloor (1943) noted that Trichomonas gallinae lost its virulence for pigeons following long-term in vitro cultivation. Gloor stated that this lost virulence could be partly recovered by the passage of the trichomonads through squabs. Meleney, Frye, and Leathers (1939) felt that long-term cultivation had not been shown to deprive completely any strain of Entamoeba histolytica of its virulence for kittens. The freezing and maintenance of protozoan cultures at subzero temperatures were initiated in an attempt to avoid the chores and consequences of long-term in vitro cultivation. In general, the results have been successful. Manwell (1943) and Wolfson (1945) maintained various species of bird malaria at temperatures as low as -78 C, and for periods up to 1 year. They noted only mild changes in infectivity of the thawed organisms from the controls, and Wolfson reported restored virulence following a few animal passages. Jeffery and Rendtorff (1955), using three malaria species from man, Received for publication 3 September 1963. * This investigation was supported by Research Grant AI-00742 from the National Institute of Allergy and Infectious Diseases, Public Health Service. froze sporozoites and asexual stages to about -70 C and kept them for over a year. The thawed sporozoites showed only slightly longer prepatent periods than those derived from mosquito bites. Weinman and McAllister (1947) maintained a variety of protozoa at -70 C, finding that some of the thawed organisms successfully infected laboratory animals after 20 months freezing. They failed, however, in their experiments with Entamoeba histolytica. On the other hand, Diamond, Meryman, and Kafig (1961, 1963) successfully cultured this ameba (as well as certain trichomonads and trypanosomatids) following its freezing and holding in liquid nitrogen (-196 C) up to 14 months. Much of the work on the freezing and subzero maintenance of trichomonads has been done by McEntegart (1954-Trichomonas vaginalis, T. gallinae, Pentatrichomonas hominis, Tritrichomonas foetus); Levine and Marquardt (1955-T. foetus); Levine and Andersen (1961-T. foetus); Levine, Andersen, Losh, Notzold, and Mehra (1962-T. foetus); Honigberg and King (1962-T. vaginalis, T. gallinae); and Diamond, Meryman, and Kafig (1963-T. vaginalis, T. gallinae, P. hominis, T. foetus). The results indicate that the quick-

Acquired immunity to migrating larvae of ascaris suum induced in pigs by repeated oral inoculations in infective eggs.

Abstract: Active immunity against migrating larvae of Ascaris suum developed in specific pathogenfree and conventional pigs following repeated oral inoculations of infective eggs of A. suum. Older pigs developed better immunity; pigs that were 2 weeks old when receiving the first oral inoculations averaged 26,068 larvae in their lungs after challenge compared with 58,926 in controls. Pigs receiving their first inoculation when 6 weeks old averaged 3,462 larvae in their lungs compared with 49,982 in the controls. Pigs developed better immunity when they received more than three egg inoculations. Four-month-old pigs that were exposed briefly to ascaris eggs on the floor of a contaminated room, then later given three additional inoculations, had no larvae in their lungs after challenge while the controls averaged 6,573. Herd pigs that had been exposed to eggs of A. suum since birth were brought to the laboratory and given six additional inoculations ranging from 20,000 to 200,000 eggs. No larvae were found in their lungs compared with an average of 38,132 in lungs of the controls. Serum gamma globulin increased in the pigs as immunity developed. Eosinophilia developed following the second inoculation but did not increase in pigs that appeared to be completely immune. There is a possibility that Ascaris suum infection in pigs can be controlled by immunizing methods such as are used against other swine diseases. Acquired immunity was investigated by Roberts (1934), who fed ascaris eggs to two pigs every day for 100 days. Typical lesions were present at necropsy but no migrating worms were found. He concluded that an immunity had developed. Controlled studies of immunity to A. suum in pigs were impossible until specific pathogen-free (SPF) pigs were developed (Young et al., 1955) because infection with ascaris could not be prevented in the experimental animals. Taffs (1958) studied active immunity to migrating larvae of A. suum using SPF pigs that had not been exposed to ascaris eggs prior to the experimental inoculations. The following report confirms the findings of Taffs and the conclusions of Roberts (loc. cit.). MATERIALS AND METHODS SPF pigs were used in three of the experiments reported herein. SPF pigs are delivered by hysterectomy, maintained in individual isolation chambers, and fed a diet of cow's milk fortified with eggs, vitamins, and minerals (Young et al., loc. cit.). They are free of ascariasis and other diseases and lack passive maternal immunity. Pigs in the fourth experiment were from a conventional herd and had been previously exposed to ascaris. The principals and controls of each experiment were of the same age, housed in separate concretefloored rooms, fed and cared for in the same

Coccidial schizonts in mesenteric lymph nodes of sheep and goats.

Abstract: Coccidial schizonts were found in enlarged mesenteric lymph nodes of sheep and goats that, 13 to 18 days previously, had been fed sporulated oocysts of Eimeria arloingi, E. faurei, and E. ninaekohlyakimovi of ovine origin. The schizonts in the lymph nodes of the goats were somewhat abnormal in appearance and measured about 32 to 100 ,u in diameter; those in the lymph nodes of the sheep were more normal in appearance and measured about 30 to 360 ,u. Mature merozoites were not found in schizonts in the lymph nodes of either the sheep or the goats; merozoites that appeared to be approaching maturity were found in the larger schizonts in the lymph nodes of sheep. Enlargement of the lymph nodes was considered to be caused primarily by edema fluid. This report concerns the occurrence of coccidial schizonts in histologic sections prepared from enlarged mesenteric lymph nodes of sheep and goats in 1959 and 1960. These animals had been fed mixtures of sporulated oocysts of ovine origin of Eimeria arloingi, E. ninaekohlyakimovi, and E. faurei during investigations of the behavior of sheep coccidia in goats. MATERIALS AND METHODS The animals from which the mesenteric lymph nodes were taken for examination were two 2month-old goats, inoculated in 1959, and two goats and two sheep, 7 months old, inoculated in 1960. Each was obtained at the time of birth, kept under strict sanitary conditions, and bottle fed until old enough to consume solid feed. They were a part of a group of 70, all of which had been raised free of helminths and were either free of coccidia or had extremely light infections of either Eimeria arloingi or E. ninaekohlyakimovi, both of unknown source. At the time of inoculation, the 2-month-old goats weighed 22 and 23 lb, respectively, the two older goats weighed 51 and 38 lb, respectively, and each of the sheep weighed 43 lb. The experimental coccidial infections were produced by placing oocyst-bearing material from pooled cultures of oocysts of ovine origin in gelatin capsules and administering them per os by means Received for publication 30 September 1963. * Present address: Veterinary Medical Division, Food and Drug Administration, Beltsville, Maryland. t Present address: School of Public Health, Johns Hopkins University, Baltimore, Maryland. of a balling gun. This material was from cultures in plain tap water and in dichromate; the cultures were 2 to 5 months old. The inocula contained oocysts of three species in about the following percentages: Eimeria arloingi, 70; E. ninaekohlyakimovi, 25; and E. faurei, 5. Expressed in millions, the approximate total number of oocysts administered to each animal was: goat 382, 60; goat 386, 2; goat 437, 0.7; goat 449, 1.4; sheep 344, 0.7; and sheep 352, 0.7. One of the 2-month-old goats, no. 382, died on the 13th day after inoculation, and the other, no. 386, was killed at that time; one mesenteric lymph node was taken from each animal. Of the 7-month-old animals, goat no. 437 was killed on the 16th day after inoculation and one lymph node was taken; goat no. 449 was killed on the 17th day after inoculation and two lymph nodes were taken; sheep no. 352 was killed on the 16th day after inoculation and two lymph nodes were taken; and sheep no. 344 was killed on the 18th day after inoculation and one lymph node was taken. The lymph nodes were fixed in buffered 10% formalin and histologic sections, about 6 ,u in thickness, were made and stained in Harris's hematoxylin and eosin-y.

Mating of ancylostoma caninum in relation to blood loss in the host.

Abstract: By oral inoculation of juvenile or adult worms, unisexual or bisexual Ancylostoma caninum infections were established in 39 young dogs. Less than 100 adult females caused rapid blood loss and anemia in two dogs at the time when the worms began laying infertile eggs. Equally heavy all-male or balanced male-female infections did not cause detectable anemia. A higher percentage of males remained in recipient dogs when transferred with (62%), than without (49%), females, but females became established equally well with or without males (average 55%). The average rate of laceration (per 100 worms) was greatest in all-female (147) and unbalanced infections (97 and 134), least in all-male infections (21), and relatively low but highly variable in balanced infections (57, range 3 to 122). Laceration rate was not related to overall population density, but when worms were very unequally distributed as they often were in all-female infections, or bunched as they were when males congregated around receptive females, the focal laceration rates were high. Blood in the worm's intestine was evident more frequently in females than in males, being seen in 53, 64, and 80% of females in balanced, unisexual, and unbalanced infections, respectively. The corresponding rates for males were 26, 9, and 37%. More than half of the worms of both sexes were unattached to the intestinal mucosa when observed 15 to 30 min after death of the host through Nembutal injection into the heart. The observations suggest that the female of A. caninum normally mates at intervals of 3 weeks or less, that mating behavior is attendant with laceration of the intestinal mucosa, especially when the sexes are highly unbalanced, and that mating may rank high among those activities of the hookworm which cause blood loss and anemia in the host. It is generally accepted that hookworms cause anemia and that the anemia results directly from blood losses caused by the worms. In the freshly opened intestine of infected dogs, blood is often seen oozing from the lacerated mucosa and filling the intestine of the hookworms. Estimates have been made of the amounts of blood passing daily through the intestine of hookworms (Wells, 1931) and the daily losses of blood into or from the intestine of the host (Roche, 1957a, b; Foy et al., 1958; Clark et al., 1961). The rate of blood loss and the consequent anemia are, in general, proportional with the number of worms present in the intestine. In extreme cases this relationship is clear and indisputable. In some instances, however, the Received for publication 6 December 1963. * Supported by Grant AI-04919 from the National Institutes of Health, U. S. Public Health Service. t Department of Medical Zoology, Kyoto Prefectural University of Medicine, Kawara-machi, Kamikyo-ku, Kyoto, Japan. relationship between worm burden and blood loss or anemia is not evident, and the lack of such a relationship cannot always be explained on the basis of duration of infection or nutriional status of the host (Foy and Kondi, 1960, 1961). The present study was undertaken to examine the possible role of sex and mating behavior of Ancylostoma caninum in the causation of blood loss in the dog. MATERIALS AND METHODS Thirty-nine mongrel dogs 3 months to approximately 6 months of age were caged separately, given a ration of commercial canned dog food, treated for helminths until stools were negative (by zinc sulfate flotation) for a period of at least 15 days, and given infections with adult or adolescent worms of predetermined number and sex. Adult worms carefully removed from freshly killed, naturally infected dogs, or adolescent worms 7 to 11 days of age, similarly taken from experimentally infected dogs, were examined individually under suitable magnification to determine the sex. Counted numbers of the worms were then transferred to 1-ml gelatin capsules and placed over the glottis of a recipient dog. Usually the capsules were

A comparison of the development of nippostrongylus brasiliensis in germ-free and conventional mice.

Abstract: Germ-free and conventional mice of similar age and sex were exposed to filariform larvae of Nippostrongylus brasiliensis treated with sodium hypochlorite and antibiotics in four experiments, and in one experiment to filariform larvae which had been given a series of sterile saline washes. In two experiments involving sodium hypochlorite-antibiotic-treated larvae the mice retained their bacteriafree status while contamination occurred with single bacterial species in each of the other three experiments. Fewer adult worms were subsequently recovered from the germ-free and monocontaminated mice than from the conventional controls in all five experiments, and in three experiments these differences were statistically significant. The number of larvae observed in lungs examined 48 hr and 8 days after infection was similar in the germ-free, monocontaminated, and conventional mice, which was interpreted to indicate that the migration of Nippostrongylus takes place similarly through the lungs of both types of hosts, but that a normal bacterial flora in the host can favorably influence the development of adult worms. The use of germ-free host animals to study host-parasite relationships, although a relatively new and little-used technique, is generally regarded as a useful system to investigate the ecology of parasitism. Phillips and Wolfe (1959), Newton et al. (1960), and Doll and Franker (1963) have used germ-free animals to study protozoan infections and Newton et al. (1959, 1962) have successfully produced helminth infections in germ-free mice and guinea pigs. These reports demonstrate the practicability of using gnotobiotic host animals and also indicate that the bacterial flora of the host may influence the development of parasites. The interest generated by these investigations, together with recent improvements in equipment (Trexler, 1959, 1963), decreasing the expense of experimentation with gnotobiotic Received for publication 20 June 1963. * From the Department of Veterinary Science, University of Wisconsin, Madison, paper NS389. This investigation was supported in part by a Public Health Service Fellowship (EPD-15,937) from the National Institute of Allergy and Infectious Diseases, Public Health Service, and in part by the American Cancer Society Institutional Research Grant to the University of Wisconsin. animals, brought about the use of these techniques in this laboratory. In initiating work with germ-free animals at the University of Wisconsin the development of Nippostrongylus brasiliensis (Travassos, 1914) in germ-free and conventional mice was studied to investigate the effect of the absence of a normal bacterial flora upon this hostparasite relationship. The particular system was chosen because germ-free mice were commercially available locally and N. brasiliensis, which has been studied extensively (Haley, 1961, 1962), appeared to be well suited for such experimentation. The in vitro studies of this parasite by Weinstein and Jones (1956, 1959) and the previous work with N. brasiliensis in germ-free guinea pigs by Newton et al. (1959) also made this system particularly attractive for these initial studies. MATERIALS AND METHODS Procurement and maintenance of animals The animals used were germ-free and conventional ARS/ICR strain random-bred albino mice and ARS strain random-bred albino rats, supplied by a local commercial laboratory animal producer (the A. R. Schmidt Co., Madison, Wis.). For each experiment the producer assembled 13 germ-free

Studies on helminths of north carolina vertbrates. v. parasites of the mink, mustela vison schreber.

Abstract: Twenty-one species of helminths were recovered from 120 mink, Mustela vison, in North Carolina. Ninety-nine hosts harbored one or more species which were grouped as follows: nine trematodes, two cestodes, eight nematodes, and two acanthocephalans. The mink is reported as a new host for three species, and North Carolina as a new locality for five species of the helminths. There are few comprehensive reports on the helminths of mink, Mustela vison. This is especially true for wild mink because many studies have been concerned only with fur farm animals. Nine species of helminths were listed from mink in Ontario, Canada (Law and Kennedy, 1932). Observations were made on the larger helminths of 158 mink in southern Michigan (Sealander, 1943). The most comprehensive report is that of Erickson (1946), who examined 151 mink of which 72 were fur farm animals. Erickson also included most of the information known at that time on helminths of Mustelidae. Additional scattered records are available but most deal with particular parasites (Ameel, 1934, 1938; Skinker, 1932, 1935; Wallace, 1935; McIntosh, 1936) or surveys which include the mink as a part of a larger study (Senger and Neiland, 1955; Voge, 1955; Freeman, 1956). This paper is concerned with the incidence of helminths in 120 wild mink in North Carolina and summarizes the current literature. The mink used in this study were obtained from commercial trappers in the Piedmont and coastReceived for publication 28 July 1964. * Contribution from the Zoology Department, North Carolina Agricultural Experiment Station, Raleigh. Published with the approval of the Director of Research as Paper No. 1846 of the Journal Series. t Supported by Public Health Service Research Grants AI-03209 and AI-05927-01, from the National Institute of Allergy and Infectious Diseases. al counties of North Carolina with the majority of them being taken in the vicinity of Raleigh, North Carolina. RESULTS AND DISCUSSION Twenty-one species of helminths were recovered from 99 of 120 mink. These included nine trematodes, two cestodes, eight nematodes, and two acanthocephalans (Table I).

Trypanosomes and other microorganisms from panamanian phlebotomus sandflies.

Abstract: One Panamanian species of sandfly, Phlebotomus vespertilionis, usually associated with bats, has a high overall rate of infection (67.9%) with a trypanosome. Both crithidial and trypanosomal forms are found in the mid- and hindgut but not in the foregut or mouthparts. In the hindgut there may be large numbers of the dominant crithidial forms attached to the epithelium. It has not been determined whether or not the sandfly trypanosome is the same as that occurring in associated bats. Trypanosomes were also found in another species, P. trinidadensis, but at a much lower rate (3.3%). Occasional infections of the hemocoel with gregarines, fungi, and nematodes have been noted in a number of species. During an extensive series of dissections of wild-caught Panamanian Phlebotomus with the primary objective of determining the extent and kind of leptomonad flagellate infections in guts of wild sandflies, a variety of other organisms was encountered. The leptomonad flagellate infections have been discussed by Johnson, McConnell, and Hertig (1963). The purpose of the present paper is to give our observations on the other organisms found in and on wild sandflies of our series. Other investigators have found a number of organisms aside from leptomonad flagellates associated with Phlebotomus, including mites, fungi, tapeworm larvae, nematodes, gregarines, and trypanosomal and crithidial flagellates (among others, Adler and Mayrink, 1961; Her- rer, 1942; Lewis and Minter, 1960; Shortt and Swaminath, 1927; Subramaniam and Naidu, 1944). In our series we found all except tape- worm larvae. Of special interest are the tryp- anosome infections and the bulk of our dis- cussion will deal with these. Trypanosomes

Properties of toxoplasma lysates toxic to rabbits on intravenous injection.

Abstract: The intravenous injection of a toxoplasma lysate into rabbits caused death in 6 to 24 hr. The toxic material has properties similar to sarcocystin and Eimeria toxin. Rabbits with chronic toxoplasmosis showed resistance to the lethal effect of the lysate. In the process of preparing hyperimmune sera in rabbits against the toxoplasma hemagglutination antigen (Jacobs and Lunde, 1957) by intravenous injection of the crude toxoplasma lysate, we noticed that these rabbits were dying within 24 hr after inoculation. This observation suggested the possibility of a toxin-like substance in the lysate. Our studies show that this substance is different from "toxotoxin" (Weinman and Klatchko, 1950) and has properties similar to sarcocystin (see review by von Brand, 1952) and a coccidial toxin described by Burns (1959).

Permeation of amino acids in moniliformis and macracanthorhynchus (acanthocephala).

Abstract: Both sexes of Moniliformis and Macracanthorhynchus concentrate methionine against a chemical difference. The uptake of methionine by both sexes of Moniliformis is inhibited by alanine, serine, leucine, and isoleucine-these inhibitions are reciprocal. The uptake of serine and alanine is not affected by methionine in female Macracanthorhynchus; however, methionine does inhibit the accumulation of those amino acids in males of Macracanthorhynchus. The effect of a mixture of four neutral amino acids on the uptake of methionine can be predicted by the generalized equation derived by Read et al. (1963, Ann. N. Y. Acad. Sci. 113 Art. 1: 154-205). Considerable information is available concerning the entrance of potential nutritional substances into cestodes (see the review by Read and Simmons, 1963, as well as Read, Rothman, and Simmons, 1963); however, there is a paucity of such data pertaining to that process in the Acanthocephala (Laurie, 1957, 1959). Like the cestodes, Acanthocephala have no gut and it is assumed that nutrients enter through the body wall. Read et al. presented exhaustive data concerning the initial rates of entry of a number of amino acids into Hymenolepis diminuta. In that study they demonstrated that amino acids were concentrated against a chemical gradient and that there was competitive inhibition between certain groups of amino acids. Those authors further demonstrated that there was inhibition of uptake of a given amino acid in the presence of a complex mixture of amino acids and that this competition could be predicted by an extension of the Lineweaver-Burk treatment of enzyme kinetics. The following study was undertaken: (1) to obtain information concerning the permeation of a selected group of neutral amino acids into two species of acanthocephalans and (2) to Received for publication 20 March 1964. * This research was supported in part by U. S. Public Health Service Grants AI-01384 and 2E-106. t Present address: California State College, Fullerton, California. further test the application of the generalized equation of Read et al. (vide supra). MATERIALS AND METHODS Acanthocephala used in this study were obtained from two sources. Moniliformis dubius were reared in male Sprague-Dawley rats (Holtzman Rat Co.). Cystacanths of Moniliformis were obtained from laboratory-reared and infected Periplaneta americana. Rats weighing 70 to 90 g were infected with 25 12-week-old cystacanths. The hosts were maintained in groups of ten in 24by 18by 12-inch wire cages in air-conditioned quarters: Food and water were provided ad lib. until the time of autopsy at 5 weeks. Rats were killed by a blow on the head and the small intestine removed to a container of saline composed of NaCl 120 mM, KCI 4.8 mM, CaCl2 2.6 mM, MgSO4 1.2 mM, and Tris (hydroxymethyl) aminomethane: maleate buffer 25 mM at pH 7.4. The intestine was opened with scissors and the worms gently removed from the mucosa. Macracanthorhynchus hirudinaceus were carefully dissected from swine small intestines at the Houston Packing Company and transported to the laboratory in the above saline in thermos containers at 36 to 39 C. Both species of parasites were separated according to sex and treated separately throughout the remainder of the experiments. The worms were preincubated in 30-ml beakers in the above saline for 60 min at 37.5 C. Following the preincubation period the worms were blotted and incubated in 30-ml beakers containing the amino acid solution (10.0 ml for Macracanthorhynchus, 5.0 ml for Moniliformis) for 3 min. The various C14 amino acids were used at a specific activity of 0.6 ,uc/,mole. After 3 min (-+5 sec) incubation, the worms were removed, rinsed rapidly in three changes of the saline (50 ml each), blotted, and placed in tubes containing a