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Reproduction and survival are influenced by parasites, and recently, the ability of the immune system to fight infection has been included in life history theory. A condition dependent immune response predicts tradeoffs within the immune system and condition dependent tradeoffs with other life history traits. Female red jungle fowl (Gallus gallus) parasitized with an intestinal nematode (Ascaridia galli) had a higher concentration of eosinophils than controls, and parasitized and control females responded differently to injections of phytohemagglutinin (PHA). PHA injections stimulated an increase in granulocytes in parasitized females relative to controls. Although parasitized females had greater circulating levels of the white blood cells that are involved in the cutaneous hypersensitivity response, control females exhibited greater swelling of the wing web six hours after injection. A. galli seemed to slow the cellular immune response to injections of PHA. suggesting a tradeoff within the cellular component of the immune system. Parasitized and control females differed in the relationship between immunoglobulin G (IgG) and the strength of the cutancous hypersensitivity response. The decrease in cutaneous hypersensitivity with increasing IgG in parasitized vs control females suggests a condition dependent tradeoff between the cellular and humoral components of the immune system.

1. Repeatability is important in determining how traits are affected by selection, but it is largely unstudied for most physiological characters other than locomotory performance. We examined the repeatability of aerobic capacity, measured as maximum O 2 consumption (Vo 2max ), in Red Junglefowl, Gallus gallus. Vo 2max is an integrated index of metabolic performance that can be intuitively linked to fitness because it sets the upper limit to sustainable power output. 2. We used mass residuals to determine Vo 2max repeatability in adult birds, and across growth from 28-day-old chicks to reproductively mature adults. We measured Vo 2max during brief episodes of intense exercise in motorized running wheels. Minimal resting metabolism (Vo 2mr ) was measured in adults to provide an estimate of factorial aerobic scope (Vo 2maX /Vo 2mr ). We also examined the influences of sex and infection by a common intestinal nematode, Ascaridia galli, on Vo 2max . 3. There were no gender differences in adult Vo 2mr or in the Vo 2max of chicks. However, the Vo 2max of adult males was considerably greater than that of adult females. Factorial aerobic scopes were 9.6 and 5.6 for adult males and females, respectively. Higher scope in males may be an adaptation to support intense, prolonged intermale aggression. Infection with A. galli significantly depressed the mass and Vo 2max of chicks but had no effect on adults. 4. The Vo 2max of adult junglefowl was highly repeatable over periods up to 180 days. In contrast, we found no repeatability of Vo 2max across ontogeny for birds tested initially as chicks and again as adults. The lack of repeatability was not influenced by sex or infection status.

SUMMARY. Three formulations of water-suspensible fenbendazole, at target doses of 30.3 or 60.6 ppm in the drinking water, were administered to broilers infected with Ascaridia galli. The medication was administered in the water through automatic medicators for 6 hours on 3 consecutive days. Three days after treatment, chickens were killed and their worms were counted. Efficacy of fenbendazole was 99.2-100% and 69.0-89.6% at administration doses of 60.6 ppm and 30.3 ppm, respectively. Worm burden and percentage of broilers infected were lower in treated broilers than in controls (P < 0.05). Formulation A was dispensed most consistently and in the highest concentration through automatic medicators into the drinking water.

A 12-month survey was conducted to determine the prevalence of poultry helminths in Northwest Arkansas commercial broiler chickens. Intestinal tracts from market-ready broilers were collected weekly from two commercial broiler companies; a total of 3542 intestinal tracts were collected for parasite enumeration from 67 company A and 52 company B farms. Ascaridia galli was found on 37.3% of company A farms and 3.9% of company B farms, Raillietina cesticillus was found on 67.2% of company A farms and 69.2% of company B farms, and Heterakis gallinarum was found on 7.5% of company A farms and 1.9% of company B farms. No other parasitic helminths were found. No significant relationship was detected between grow-out feed efficiency and helminth infection rate or magnitude.

Eggs cooled to -22 or -93 C were shrunken, and/or depressed. The topography of frozen eggs appeared abraded and mottled compared to the smooth surface of shelled, unfrozen controls. A smooth seam on the roughened lipid surface of deshelled, air-dried eggs appeared as a line of dehiscence. The folded and wrinkled lipid surface of deshelled, freeze-dried eggs had a polar ridge. The only published report of scanning electron microscopy (SEM) of nematode eggs is that of Ubelaker and Allison (1972). They described an elaborate reticular network producing irregular depressions on the surface of Ascaris lumbricoides and A. suum eggs. Also, a round depression was seen at one pole, a possible site of larval egression. In our preliminary cryobiologic studies, more fertilized, shelled eggs (chitinous layer present) survived freezing to -22 C than to -93 C. Also, 5 and 10% glycerol was toxic to fertilized, deshelled eggs (chitinous layer removed), but 5 and 10% dimethyl sulfoxide (DMSO) was not. The SEM was used to delineate effects tfiat may explain these observations. MATERIALS AND METHODS Shelled eggs were recovered from female worms incubated at 38 C in petri dishes containing 0.85% NaCi solution. These eggs were gently dislodged from the bottoms of petri dishes, pipetted into clinical centrifuge tubes, and washed 5 times with distilled water. Shelled eggs were suspended in distilled water and sealed in 1.2-cc glass ampules. Ampules were placed in either a refrigerator freezer (ambient temperature reached -22 C in approximately 30 min) or a REVCO low-temperature freezer (ambient temperature reached -93 C in approximately 9. min) for 24 hr. After thawing at 38 C, the shelled eggs were treated as follows: Fixed for 30 min at room temperature in 12% glutaraldehyde in 0.05 M Sorensen's phosphate buffer, pH 7.2; washed several times in 0.05 M Sorensen's phosphate buffer, pH 7.2; postfixed for 30 min at room temReceived for publication 17 August 1973. * Contribution No. 1208, Division of Biology, Kansas Agricultural Experiment Station, Kansas State University, Manhattan, Kansas 66506. The authors acknowledge technical assistance of Dr. Charles Pitts and Mr. Paul Enos. perature in 1% osmium tetroxide in 0.05 M Sorensen's phosphate buffer, pH 7.2; washed several times with deionized, double-distilled water. Shelled eggs maintained at 30 C for 24 hr (controls) were fixed in like manner. All shelled eggs were then mounted on aluminum stubs and dehydrated as follows: A thin layer of ethyl acetate extract of double-coated tape No. 666 (3M Co.) was applied to the stubs; a small drop of deionized, double-distilled water containing eggs was pipetted at intervals around the edge of the stub; the stubs supporting the specimens were placed in a desiccator and dehydrated by air-drying overnight. Then dehydrated specimens were removed and coated with a thin layer of gold and palladium. Eggs were deshelled to expose the lipid layer according to the method of Elliott (1954). Five groups of deshelled eggs were placed one group each into 5 and 10% concentrations of dimethyl sulfoxide or glycerol and in distilled water (as controls). All tubes of eggs were rotated on a roller drum apparatus at ?/-. rpm for 12 hr at 23 C. The deshelled eggs were fixed, washed, mounted on stubs, and air-dried the same as the frozen shelled eggs were. In addition, a second group of mounted deshelled eggs were freeze-dried according to the method of Weise (1973). Specimens were examined with the ETEC scanning electron microscope (ETEC Corp.) at an accelerating voltage of 20 kv.

Broiler chickens were fed ad libitum diets containing fenbendazole at various concentrations and durations for the removal of adult Ascaridia galli, Heterakis gallinarum, and Capillaria obsignata. All regimens were 100% effective against A. galli. Fenbendazole at doses of 80 ppm for 3 days and 48 or 30 ppm for 5 days was 100% effective against H. gallinarum. C. obsignata was not removed completely by any regimen, but greater than or equal to 95% effectiveness was seen in birds given fenbendazole at doses of 80 ppm for 3 days and 48 ppm for 5 days. All medicated rations were readily consumed, and the treated birds exhibited no adverse reactions.

SUMMARY Earthworms were found to retain eggs and larvae of Ascaridia galli very briefly, and transmission can be attributed only to such eggs or larvae in transit in the digestive tract. Larvae of A. galli do not accumulate as do the larvae of Heterakis gallinarum, for the larvae of A. galli are voided within 48 hr of ingestion of the eggs. Since these larvae are unprotected, they must usually die, having little opportunity to gain access to a definitive host.

In a comparative field trial, 5,950 meat breeder chickens were medicated via feed for 7 consecutive days with 60 ppm of the anthelmintic flubendazole. This treatment cured the birds of an infection with Ascaridia galli and Capillaria obsignata. A slight but transitory diarrhea was observed in treated chickens. The applied medication did not unfavorably affect egg production, fertility, or hatchability.

White Leghorn chickens were obtained from a commercial hatchery as day-old birds, vaccinated against Newcastle disease, and fed an 18% protein antibiotic-free ration throughout the experimental period. At 14 days of age the birds were banded, weighed, and divided into groups of equal weight according to the method of Gardiner and Wehr (3). At that time, birds to be infected in Trials 1 and 3 were given 100 ?+ 10 larvated eggs (4) of A. galli, and birds in Trials 2 and 4 received 75 ? 10 larvated eggs of H. gallinarum. Fewer than 100 eggs of cecal worms were used, so as to reduce the likelihood of histomoniasis. Nevertheless, laparotomies were performed on these birds 10 days postexposure to cecal worm eggs in order to eliminate from the efficacy tests all birds showing cecal cores (histomoniasis). Fifty-eight days postexposure to larvated eggs, efficacy tests were initiated according to the experimental design and drug regimens given in Table 1. Because the two drugs were insoluble in water, accuracy in dose was obtained by keeping them in suspen

Larval stages of Ascaridia numidae in guinea fowls did not migrate into the liver, lungs, or other internal organs of the host. Larvae were found associated with the mucosal surface of the jejunum and ileum walls on the 4th to the 12th days of the infection by use of the Baermann technique. Before and after this period larvae located primarily in the lumen of the jejunum and ileum. Survival and development was similar in germfree and in guinea fowls which had conventional flora. The heavy parasite burden of A. numidae found in semiwild guinea fowl in Georgia has not been previously reported. Although the life cycle of Ascaridia numidae (Leiper, 1908) has recently been described as being direct (Barus, 1968; Macchioni, 1971), studies on larval tissue phases of the life cycle are incomplete. Ascaridia columbae (Wehr and Hwang, 1964) commonly invade the liver and lungs of pigeons. Two other species, Ascaridia galli (Ackert, 1931; Edgar et al., 1957) and A. dissimilis (Horton-Smith et al., 1968), rarely migrate to the visceral organs in the chicken or turkey. Disagreement is recorded on the extent and significance of larval penetration into the mucosa of the chicken (Ackert, 1923; Tugwell and Ackert, 1952; Todd and Crowdus, 1952; Moran and Mizelle, 1957; Madsen, 1962). Investigations of deep and mucosal tissue penetration of A. numidae, using new combinations of techniques, are herein reported. MATERIALS AND METHODS

The egg of Ascaris has been widely used as a subject for cytological investigations. Nelson (1851) described the development of the egg of Ascaris mystax. Leuckart (1886) and Wharton (1915) reported the Ascaris lumnbricoides egg to be surrounded by a shell and an outer, albuminous layer. The appearance of unfertilized eggs of this species was described by Otto (1932) and Keller (1933). The number of layers surrounding the egg of ascarids has been reported as three (Nelson, 1851, Ascaris myvstax; Leuckart, 1886, Ascaris ilumbricoides; Ackert, 1931, Ascaridia lineata; Wottge, 1937, Parascaris mnegalocephala; Christenson, 1940, Ascaris lumbricoides), four (Kreuzer, 1953, Ascaris lumabricoides; Frenzen, 1954, Ascaridia galli, and five (Zavadovsky, 1928, Ascaris). A variety of techniques has been used by different authors in studying the egg layers, including microscopic observations of the penetration of substances into the egg, polarizing and fluorescent microscopy, centrifugation, and chemical tests on the membranes. The chemical composition of the layers has been investigated by several workers (Faure-Fremiet 1913, 1913a; Chitwood 1938; Christenson, et al. 1942; Jacobs 1940; Timm 1950; Monne and H6nig 1954; Fairbairn 1955) and, in general, have led rather consistently to the conclusion that there are three membranes. Wottge (1937) described the endogenous formation of the egg layers of Ascaris megalocephala, whereas Christenson (1940) believes the outer layer of the Ascaris egg to be of exogenous formation. Morita (1953) examined paraffin sections of the egg shell of Ascaris megalocephala with the electron microscope and concluded that the layer "consists of completely individualized microfibrils." In the present investigation, thin sections were examined with the electron microscope to determine structural details of ova and the order of formation of the egg coverings of Ascaris lumibricoides var. suum.

Propylthiouracil (PTU) was employed to induce hypothyroidism in albino mice which were subsequently given 6,400 or 10,000 Trichinella spiralis larvae. During the first 8 days of infection there was no difference in the mean number of hypothyroid and euthyroid (normal) animals that died. But differences in survival times between the respective hypothyroid and euthyroid groups were statistically significant. The mean survival times of mice receiving 6,400 larvae were 79 and 82 hr for the two groups fed PTU, and 110 hr for the control group. When 10,000 larvae were used, the survival times of mice in hours were 64 and 59 for the PTU-treated animals and 106 for the controls fed a PTU-free diet. When survival time is used as a criterion, the results suggest that the hypothyroid state lowers the resistance of mice to heavy T. spiralis infections. In another experiment second generation larvae were recovered from hypothyroid and euthyroid mice 6 weeks after each received 300 larvae. Although more larvae were recovered from hypothyroid animals (37,000 per mouse) than from controls (27,000 per mouse), the difference was not significant. It is unlikely that larviposition is affected by the thyroid status of the mouse host. Studies of the relation between host thyroid activity and parasitic infections have been few, the results variable. Thus, Todd (1949) working with chickens made hypothyroid with thiouracil or hyperthyroid with iodocasein reported that Heterakis gallinae developed to a greater size in hypothyroid hosts, but Ascaridia galli grew larger in hyperthyroid birds. However, the number of nematodes recovered from chickens with either thyroid abnormality was not significantly different from those in controls. According to Dobson (1964), surgical thyroidectomy increased the number of Oesophagostomum columbianum surviving in lambs, particularly female lambs. Thyroidectomy had no effect, however, on the number of nodules found in hosts of either sex. Using the number of cysticercoids observed 93 hr after infection as the criterion of resistance, Larsh (1947, 1950) reported that the hyperthyroid condition favored the development of Hymenolepis nana in mice. No effect was noted in thiouraciltreated hypothyroid hosts. On the other hand, Landt and Goodchild (1962) recovered heavier Received for publication 11 July 1966. * This investigation was supported by NIH Research Grant NBO 1716 awarded to Dr. M. Hamburgh. t This project was carried out with the assistance of Mr. F. Weil, Rutgers University, and Miss D. Gelfand, Brooklyn College, who participated under the terms of an NSF Summer Institute Grant awarded to Yeshiva University. specimens of H. diminuta from thyroidectomized female, but not male, rats. Palva (1963) detected no relation between hypothyroidism and impaired absorption of vitamin B12 in patients infected with the broad tapeworm, Diphyllobothrium latum. Wheeler et al. (1948) found that hypothyroidism did not alter the natural resistance of thiouracil-fed chicks to Eimeria tenella. Differences in technique and biology of the parasites may account for the diversity of these results. The need for more information on the relation between thyroid activity and parasitism stimulated the present work. The experiments reported herein were undertaken to determine whether hypothyroidism in mice would affect (1) the survival of mice infected with massive doses of Trichinella spiralis, and (2) the number of second generation larvae recovered from host tissues when nonlethal infections were given. MATERIALS AND METHODS The mice used in these experiments were male Swiss albinos from the Charles River Breeding Laboratories. The animals ranged between 5 and 6 weeks of age at the start of each experiment. They were tagged and housed in individual metal cages on wood shavings. The cages were cleaned and new wood shavings added every week. During the experiments all animals were maintained on powdered Rockland mouse diet. The food was issued in porcelain bowls and changed every day. The animals were divided into three groups. Group A consisted of mice that were offered a

Knowledge of the rate of development, resistance, and viability of nematode eggs has been accumulating for nearly a century. Most of the work has been concerned with the ascarids of pig and man, but some studies have been made on the rate of development and viability of the eggs of Ascaridia galli (Schrank, 1788). McRae (1935) reported that the ova of A. galli did not develop beyond the tadpole stage when kept in an atmosphere 40 to 50 per cent saturated, and only an occasional ovum developed beyond the morula stage at this relative humidity. Ova kept in 82 to 86 per cent relative humidity at 220 C. developed more slowly than those in water, whereas ova kept in 100 per cent relative humidity at 300 C. developed at the same rate as did those in water. Ackert (1931) reported the optimum developmental temperature for A. galli to range from 300 to 330 C. Twelve hours exposure to 430 C. proved to be lethal for these ascarid eggs in all stages of development. Such environmental factors as temperature, shade, and depth below surface of the ground influenced the development and survival of A. galli ova (Ackert and Cauthen, 1931; Levine, 1937). Age of infective ova is also a factor in their viability. Ackert et al. ('1947) reported that egg cultures of A. galli incubated in water at 300 to 330 C. for 36 days proved to be more viable than eggs of a 120-day-old cul-ture. Their criterion for judging viability was the average lengths of the worms recovered from experimental chickens subsequent to feeding eggs from the two types of cultures. Todd et al. (1950) used the criterion of failure on the part of the host to gain weight subsequent to infection with A. galli as an indication of virulence oif the nematodle, whereas an increase in worm numbers accompanied by an increase in host weight indicated optimum viability of the infective larvae. They reported that the greatest host injury resulted from feeding chickens infective eggs 14-21 days old that the virulence of the parasite decreased as the infective eggs aged. The present paper reports the results of experiments designed to compare the rates of development, viability, vigor, and virulence of larvae developing from A. galli ova cultured respectively in 90 per cent relative humidity and in shallow water.

A new antiparasitic agent, 3-methyl-5-[(p-nitrophenyl)azo]rhodanine, "Nitrodan," was effective when administered in feed against Hymenolepis nana and Syphacia obvelata infections in mice, Ascaridia galli infections in chickens, and Toxocara canis, Ancylostoma caninum, and Uncinaria stenocephala infections in dogs. This drug was not effective against Aspiculuris tetraptera, Nematospiroides dubius, and Nippostrongylus muris in mice or Toxascaris leonina in dogs. Nitrodan fed for 21 days at levels of 250 to 1,000 ppm produced significant destrobilization of H. nana in mice but removed few of the scolices. Levels of 500 to 1,000 ppm nitrodan fed for 21 days were 86 to 90% effective in reducing the numbers of adult and immature S. obvelata in mice. Nitrodan administered at 250 and 500 ppm for 4 weeks against artificial laboratory infections of A. galli was 59 and 94% effective. Comparable levels of 230 and 460 ppm nitrodan fed for 6 weeks or longer under field conditions were 94 to 100% effective which indicated lengthening the feeding period increased efficacy. In dogs, nitrodan exhibited activity against T. canis, A. caninum, and U. stenocephala by reducing egg production prior to promoting gradual elimination of the worms. Nitrodan levels of 100 to 500 ppm rapidly reduced T. canis egg production and worm burdens, and produced efficient, but not complete, removal of both hookworm species in 4 to 8 weeks. A method of initial drug evaluation using five species of helminths in mice is also presented. Recent reports of efficacy of relatively longterm feeding of anthelmintic drugs to rats (Cuckler et al., 1965), dogs (Wood, et al., 1965), poultry (Boisvenue and Hendrix, 1965), swine (Isenstein and Todd, 1965), and cattle (Cox et al., 1965) suggest this method of controlling parasitism may prove more efficient in animal production than individual dosage administration (s). Candidate compounds for continuous feeding require maximum tolerance by the host but theoretically offer maximum opportunity for antiparasitic effect. The mechanisms involved for chemoprophylactic elimination of helminth populations may be quite different from the more conventional vermifuge action sought by single or intermittent drug administration. Nitrodan is representative of a new class of antiparasitic drugs. CH:--N-C=O I S=C C-N=N7 -NO2

The literature is replete with references to a greater resistance in older animals than in younger ones against the establishment of nematode parasites. Experimental data confirming the existence of age resistance have been presented for a variety of nematode host systems: viz, Ancylostoma braziliense in cats and dogs (Sarles, 1929) ; Nippostrongylus muris in rats (Graham, 1932 and Chandler, 1932); Strongyloides ratti in rats (Sheldon, 1937); Trichinella spiralis in rats (Nolf and Zaiman, 1941); and Ascaridia galli in chickens (Ackert, Porter and Beach, 1935). On the other hand, there was no evidence of age resistance to Trichostrongylus calcaratus in rabbits (Sarles, 1932) or to T. colubriformis in guinea pigs (Herlich, 1958). Although there has been considerable reference to age resistance against nematode parasites in ruminants, evidence of this phenomenon is largely empirical being based primarily on field observations of naturally infected cattle. Under these circumstances, varying degrees of acquired immunity cannot be ruled out as a factor in the causation of the observed facts. Experimental data have been presented by Lucker (1952) and Goldberg (1952) suggesting that older sheep are somewhat more resistant than younger ones to infection with Haemonchus contortus and Oesophagostomum venulosum. However, Gordon (1950) stated that "age resistance appears to be practically absent as far as haemonchosis is concerned" in sheep Furthermore, Stewart and Gordon (1953) reported that 7 of 10 threeto four-yearold sheep raised worm-free died from experimental exposure to T. colubriformis. Host age appeared to have no influence on susceptibility to infection with 0. radiatum and Bunostomum phlebotomum in calves ranging from 0.5 to 1.25 years old (Mayhew, 1940) or to Cooperia punctata in cattle up to 1 year old (Bailey, 1949). On the other hand, the course of infections of calves with Strongyloides papillosus was affected by age of the host (Vegors, 1954). In rather striking contrast, however, various investigators (Bailey and Herlich, 1953; Andrews et al, 1953; and Bailey and Thorson, 1954) have incriminated gastrointestinal nematodes as the cause of severe clinical parasitism and deaths in adult cattle. As there is a paucity of unequivocal experimental evidence of age resistance to nematode parasites in cattle, some experiments were conducted in an attempt to clarify the status of this phenomenon; the results are herein reported. Some of the data from one of the experiments have been previously published in abstract form (Herlich, 1956b).

Studies on the development of Ascaridia galli from X-irradiated embryonated eggs indicated that a complete absence of male worms is due to their elimination rather than to sex reversal. Elimination of males occurred no later than 10 days postinfection, the earliest when worms could be sexed by current methods. Results of hatching X-irradiated embryonated eggs in vitro indicated that under the conditions used potential male larvae were capable of freeing themselves from the eggshell. Absence of male worms after experimental infection of chickens with X-irradiated (5 to 80 kr) embryonated (infective) eggs has been reported (Varga, 1964; Ruff, Hansen, and Ostlind, 1965). Reports on other nematodes revealed a change in sex ratio but no complete elimination of males at low levels of radiation (5 to 20 kr). The purpose of this study was to determine if the absence of males was related to selective elimination of males or to sex reversal, and when the effect occurred. Sex reversal was hypothesized because physical factors such as temperature (Yoshikura, 1959) and chemical substances (hormones) (Hishida, 1965) have been shown to result in sex reversal in vertebrates. Radiation-induced sex reversal has been reported in a plant (Gomez-Campo and Casas-Builla, 1965). MATERIALS AND METHODS Eggs of Ascaridia galli were incubated (2 weeks) until they contained infective larvae. Some eggs were exposed to 10 kr of X-ray using a 150 kv Picker unit (tungsten target) while others served as nonirradiated controls. The techniques of measurement of dosage level and rate were those of Ostlind and Hansen (1966). Sixty-two, 7-day-old white rock chicks were each given per os 200 ? 20 irradiated or nonirradiated eggs according to experimental design. Intramuscular injections with 0.625 mg hydrocortisone acetate were given birds on the day of exposure to eggs and continued on days 3, 6, 9, 13, and 16 postexposure in order to increase worm burdens for better test comparisons. In two experiments birds were killed at various days postexposure (Fig. 1), and all lumen dwelling worms were recovered by flushing the intestines according to the method of Ackert and Nolf (1929). Received for publication 26 June 1967. * Contribution No. 401, Division of Biology, Kansas Agricultural Experiment Station. Supported by AEC Contract No. AT(11-1)-1209, publication COO-1209-5. Also, each intestine was slit and shaken vigorously in water to remove any larvae attached to the intestinal mucosa. All flushed and washed contents were combined and examined for worms using a dissecting microscope. The slit intestine was also examined to insure that all larval A. galli had been removed. Ruff et al. (1965) using the technique of Herlich (1956) showed by digesting the chicken intestine that the aforementioned methods were effective for recovering worms. Male worms were distingushed from females either by the presence of a preanal sucker (16to 65-day-old A. galli) or by the presence of a preanal swelling (10to 15-day-old worms) according to Ackert (1931). Worms younger than 10 days old showed no reliable character by which they could be sexed. Possibly, X-irradiation merely hindered hatching of potential male larvae. Studies on in vivo hatching of irradiated embryonated eggs were precluded because the small size of A. galli at 1 day postexposure prevents recovering adequate numbers and larvae cannot be sexed before the 10th day postexposure. This factor was investigated in vitro by comparing the percentages of embryonated (irradiated and nonirradiated) eggs hatching after 24 hr incubation at 40 C in an intestinal extract obtained from 6-week-old birds. The extract was prepared from a portion of the intestine beginning at the gizzard and extending approximately 5 inches posterior to the bile duct. The contents were pressed out, centrifuged at 1,000 RPM for 5 min and the supernatant filtered twice using two thicknesses of No. 2 Whatman filter

Babero ( 1952) reported that X-irradiation of unembryonated Ascaridioz galli eggs resulted in a decrease in nutnbers becoming larvated as roentgen dosages increased. Shikhobalova, Vasilkova, Shekhtman and Vinogradova (1958) exposed eggs of Ascaris lgmbricoides, A. svgm and Ascaridia galli at various stages of development ranging from tygote to infective larvae. Using larvation as a criterion of effect, they reported that the eggs were most sensitive to irradiation at the blastula, morula and early gastrula stages. The present study was initiated to verify the report of Babero (loc. cit.), who irradiated unsegmented eggs, and was extended to study -the X-irradiation of lavated eggs, observing effects on survival, growth rate, naorphology, and sex of worms developing from such eggs. The words "significant" and "signiScance" in this report have a statistical connotation (t .05).

HIGH incidence of light infections of the intestinal roundworm Ascaridia galli commonly occurs in broiler production. This is a remarkable build-up of worm population considering the fact that the minimum life cycle requires 51/2 weeks and broilers are marketed at 9 weeks. Furthermore, the excellent balanced rations used for growing chicks has eliminated nutritional deficiencies which are important predisposing factors to heavier worm populations. A recently completed survey of broilers in north Georgia (Reid1), showed that 59 percent of 100 flocks had birds infected with roundworms at the time they were marketed. Each flock was represented by 10 bird samples. A total of 240 or 24 percent of the 1,000 birds examined was infected. The average number of worms in infected birds was 6.2. TWo flocks could be classified as heavily parasitized with 100 percent infection averaging 33.2 worms each. The other 57 were classified as lightly infected with an average of 4.3 worms each.

The resistance of ascarid eggs to varying conditions of temperature, humidity, and to various chemical agents is well known. Seamster (1950) and Jaskoski (1952) reviewed the pertinent literature and especially that concerning eggs of the swine ascarid. Ackert and Cauthen (1931) observed the effects of varied climatic conditions on the eggs of Ascaridia galli. Christenson et al. (1942) made a comparative study of the development of eggs of Ascaridia galli and Heterakis gallinae. They reported that eggs of A. galli were slightly less resistant to nicotine alkaloid than those of H. gallinae, but that Heterakis eggs had a lower thermal death point and appeared to be viable for longer periods of time. Hartman (1923) and Ackert and Cauthen (1931) observed that the eggs of Ascaridia from chickens were less resistant than the eggs of mammalian ascarids. Owen (1928), Taylor (1924), and Otto (1929) reported on the resistance of eggs of Toxacara canis and Toxacara cati under varied soil conditions and experimental laboratory conditions where temperature and humidity could be controlled. Eggs of the common cat and dog ascarids were reported to be generally more resistant to cold than to warm, dry temperatures. Lucker (1939) observed that certain haldgenated hydrocarbons were lethal to the eggs of T. canis. Chlorosol and ethylene dichloride were found effective ovicides in the soil, the latter compound being more effective than CC14 or dichloropentane. The nature of this resistance may best be sought by examination of the three egg membranes surrounding the ascarid ovum. Chitwood (1938) observed that the outer membrane is a protein, the middle membrane a chitinous supporting layer, and the inner a lipoid membrane. Ransom and Foster (1920), Zawadowsky (1928), and Hyman (1951) pointed out that the lipoid membrane is extremely important in regard to egg resistance* and is impervious to all materials except lipoid soluble ones. Since the chemical constituents of egg membranes of most ascarids are the same, it appeared timely to test the effects of chemical agents on the eggs of several of these species. The classification by Chitwood (1950), including Heterakis in the superfamily Ascaridoidea, is used here. In preliminary work the author (1950) noted that certain commercial detergents exhibited a faster ovicidal effect if the tw"o outer coats of the swine ascarid egg had been dissolved by a hypochlorite solution (anti-formin) before subjection to the detergents. Acknowledgement.-The author gratefully acknowledges a generous supply of the detergents used from the Monsanto Chemical Company, the Union Carbide and Carbon Corporation, the Allied Chemical and Dye Corporation, and the E. I. DuPont de Nemours and Company. Appreciation is also expressed to Armour and Company, Omaha, for a supply of swine ascarids; to Dr. K. B. Kerr and Mr. W. C. McGuire of Dr. Salsbury's Laboratories for generous supply of chicken ascarids and heterakis used. Miss Jean Bonnema assisted in the experimental work.

The intestinal roundworm, Ascaridia galli (Schrank, 1788), continues to present an economic problem to broiler growers in southeastern United States in spite of the ever decreasing growing period required to produce a 3-pound broiler. In recent studies (Reid, 1958) samples of 10 birds from each of 100 farm flocks showed 59 percent of the flocks and 27.5 percent of the birds carried A. galli at marketing time when birds averaged about 9 weeks of age. Large expenditures for worm treatment have proved disappointing in the amount of control thus achieved. Earlier studies (Ackert, 1931) suggested that the life cycle of A. galli required up to 50 days, but many birds are now marketed between 56 and 70 days, thus making it difficult for a life cycle of that duration to be completed. A restudy of the life history of the parasite thus seemed desirable to answer the riddle of the survival of the parasite within these short growing periods and to give basic information useful in planning control programs. Kerr (1955), in a series of experiments which were devised to establish the minimum maturing time of the worm in birds of different ages, showed that fertile eggs were recovered by fecal examinations as early as 30 days after infective eggs were ingested by 12-day-old chicks. Somewhat longer periods were required for older birds. Roberts (1937) found fertile eggs in the intestine as early as 27 or 28 days after infection with a 100 A. galli eggs. The 50-day period reported by Ackert would appear to be the average maturing time in somewhat older birds. No similar study on the minimum maturing time for eggs to develop to infectivity has been reported in the literature. Such work, together with temperature studies as related to development in the litter of brooder houses, is here reported.

The life cycle of Ascaridia columbae, the large intestinal roundworm of the pigeon, with descriptions of the preparasitic and parasitic stages, has been studied. Under conditions and methods employed the first-stage larva appears in the egg in 12 to 15 days. This larva molts to the second or infective stage in another 4 or 5 days while still within the egg. Second-stage larvae are encountered in the liver of nearly all infected birds. Periodic examination of larvae from the livers of infected birds killed every few days for 50 days after infection revealed that no development takes place in the liver. However, all stages in the development of the parasitic larvae and growth of worms to maturity were observed within the small intestine. The molt to the third stage occurs between the 3rd and 6th days after infection, to the fourth stage between the 11th and 15th days, and to the fifth stage between the 16th and 19th days. Adult worms attain a length of 40 to 50 mm. Eggs appear in the feces of the host 37 to 42 days after infection. Differentiation of the sexes is possible in the late third-stage larvae. Males are slightly shorter than females, and the precursor of the preanal sucker of the male is indicated by a swelling in the region just anterior to the anal opening. Larvae were observed in the intestinal mucosa as early as 5 hr after ingestion of embryonated eggs and as late as the 25th day after infection. Invasion of the intestinal wall by the larvae of A. columbae is discussed in the light of observations on their behavior and on that of larvae of Ascaridia galli. However, no definite decision is reached as to its relationship to the development of A. columbae. Information on the life history of ascarid nematodes of domestic birds is limited almost exclusively to that of the large intestinal roundworm of chickens, Ascaridia galli. An understanding of migration and development of other species of ascarids of these and other birds would not only augment our knowledge of these worms but would be useful for formulating effective control programs. Ascaridia columbae, a common parasite of the small intestine of the pigeon Columba livia domestica, is the only ascarid that occurs in this bird. The adults have been well described by Irwin-Smith (1920), Baylis and Daubney (1922), and Cram (1927). Unterberger (1868) reported that Ascaridia maculosa (= A. columbae) developed to maturity and eggs appeared in the feces 17 or 18 days after feeding embryonated eggs to the Received for publication 17 May 1963. 131 pigeon. Bedel (1902) observed larvae of this nematode in the liver of pigeons. Hwang and Wehr (1958) and Wehr and Hwang (1959) likewise found larvae of A. columbae in the liver as well as in the lungs of experimentally infected birds and suggested that migration through these organs might be essential for their development. The purpose of the present study was (1) to make observations on the life cycle of the pigeon ascarid, (2) to provide descriptions of the preparasitic and parasitic stages of this nematode, and (3) to determine whether migration outside the intestinal tract occurs. MATERIALS AND METHODS Infective eggs were obtained as required by immersing viable, egg-bearing female worms in 1 or 2% formalin for approximately 30 days. Ninety-one pigeons, 6 to 8 weeks old, were employed in these tests. Forty-nine of these birds were obtained from a large pigeon plant in the This content downloaded from 157.55.39.195 on Tue, 26 Apr 2016 06:09:40 UTC All use subject to http://about.jstor.org/terms 132 THE JOURNAL OF PARASITOLOGY, VOL. 50, NO. 1, FEBRUARY 1964 TABLE I. Location and relative numbers of worms recovered at necropsy from pigeons given single doses of from 500 to 1,000 embryonated eggs.1 Duration of N L Duodenum Jejunum Ileum infection o No. birds Liver infection No. blrs Llver Washings Scrapings Washings Scrapings Washings Scrapings 5 hours 1 x x 6 ,, x x 8 ,,1 x x x 14 ,, 1 x x 16 2 xx x xxxx xxxx xxxx 20 1 x 2 1 xxxx x xx x 1 day 3 xxxx x xx x 2 days 7 x x x 3 ,, 6 x x 4 3 x x 5 ,, 2 xxxx x x 6 3 7 ,, 2 x x 8 , 2 xxxx xx x 9 ,, 1 x x 10 ,, 3 xxx x x 13 ,, 4 xxxx x x x x x 14 2 x x xx x 6 ,, 2 xxx x 17 ,, 2 xx xxxx xxxx 18 ,, 1 xxx xxxx xxxx xx x xx x 25 , 3 x x xxxx x xxxx 42 , 2 xxx x xxxx xxxx 50 2 x 1 The number of x's opposite two or more birds necropsied at a particular hour or day represents the average number of worms observed. x = 1 to 10; xx = 11 to 30; xxx = 31 to 100; xxxx = 100 or more. southern United States and the remaining 42 birds from the Animal Husbandry Research Division, Agricultural Research Center, Beltsville, Maryland. All the birds were held in cages for several days or weeks before introducing them into an experiment. Prior to each experiment, the feces of each bird were examined for ascarid eggs for 4 consecutive days. Only those birds whose feces were negative for these eggs were used for experimental purposes. The female worms were first ground up by mortar and pestle to facilitate counting and feeding the eggs. Fifty-eight of these birds (Table I) received a single dose of 500 to 1,000 embryonated eggs each. The birds were killed and examined periodically from 5 hr to 50 days after infection. Seventeen other birds (Table II) were given approximately 1,000 embryonated eggs in each of 2, 4, 5, and 11 doses, the interval between doses varying from 4 hr to 24 hr in those birds which received four or more doses, and 17 hr in those administered two doses. The purpose of the multiple versus the single doses of eggs was to determine the distribution of the larvae within the body following the two methods of infection. The 16 remaining pigeons were fed approximately 1,000 embryonated eggs each and were killed and examined at 1-hr intervals for the first 8 hr and daily thereafter through the 9th day after infection. At necropsy, portions of the duodenum, jejunum, and ileum were preserved in 10% neutral formalin for sectioning. Sections were examined microscopically to determine whether the larvae had penetrated the mucosal lining of the intestine and, if so, the approximate time this invasion took place after eggs were ingested. Larvae were recovered from the experimentally infected birds by the following procedures: The lungs were removed first by lifting them through openings cut in the side of the body. They were then placed in sterilized dishes containing physiologic saline. The body cavity was flushed out with a small amount of saline that was allowed to drain into a large settling dish. Samples of blood were drawn separately from the portal vein and right ventricle of the heart into sterile hypodermic syringes containing small amounts of 2% sodium citrate solution and added to glass beakers containing saline. The liver, heart, and intestines were then removed separately, rinsed in tap water to remove any larvae adhering to the outer surfaces, and placed in separate beakers to which a small amount of saline had been added. The liver (after removal and examination of the bile duct) and lungs were cut into small pieces and pressed through a 25 mesh/inch screen. The comminuted material was collected in a beaker filled with saline. The mesenteries, along with their blood vessels, were pressed between glass plates and examined for living larvae under the dissecting microscope. The small intestine was divided into the duodenal loop, the jejunum, and ileum. Each section was opened separately and agitated in saline so as to remove all larvae that might be free in the lumen or loosely adhering to the mucosa. The walls of these three portions of the intestine were then scraped, comminuted by pressing through a finemeshed screen, and placed in beakers of warm physiologic saline. Scrapings from the trachea, esophagus, and crop were placed in separate beakers of warm saline. The contents of the beakers were stirred to break up the masses of mucus and This content downloaded from 157.55.39.195 on Tue, 26 Apr 2016 06:09:40 UTC All use subject to http://about.jstor.org/terms WEHR AND HWANG-ASCARIDIA COLUMBAE 133 TABLE II. Location and relative numbers of larvae of Ascaridia columbae recovered at necropsy from pigeons given multiple doses of embryonated eggs.1'2 Duration of No. Doses of Bile duct Portal L Jejunum Ileum infection birds eggs e uct ve Linungss) ( washings ) (scrapings) (Days after (Number) last inoc.) 1 1 2 x x 2 1 2 x x xxxx 6 1 2 xx x x xx 7 2 11, 4 x xxxx x 8 2 11, 2 x xxxx x xxxx 9 1 4 x x 10 1 2 x xxx x x 4 2 x 15 2 11, 5 xxx xxxx xxxx 16 1 5 7 1 5 x 18 1 5 xx x 9 1 5 xx x 26 1 5 x xxx x Each bird received approximately 1,000 embryonated eggs at each dose. 2 The number of x's opposite two or more birds necropsied at a particular hour or day represents the average number of worms observed. x = 1 to 10; xx = 11 to 30; xxx = 31 to 100; xxxx = 100 or more. other materials. The beakers were sedimented and decanted several times, adding fresh saline solution, and then examined for larvae.

The viscera of 262 wild ring-necked pheasants (Phasianus colchicus) were examined for helminth parasites. All of the birds were taken in Brown County, South Dakota, during various months of 1961. The collection consisted of 92 adults, 165 juveniles (young-of-the-year), and 5 birds of undetermined age. Parasitic worms were found in 97 (37.02 percent) of the pheasants, with 86 harboring only a single species of helminth. The cecal worm (Heterakis gallinarum) was the most prevalent helminth, occurring in 92 (35.11 percent) of the birds; this incidence was distributed throughout the year and was significantly higher in adult birds. The tapeworm (Choanotaenia infundibulum) was found in 15 birds (5.73 percent), but did not occur in any of the 106 birds taken during the winter. The intestinal nematode (Ascaridia galli) was found in one bird. Despite the fact that the ring-necked pheasant has long been established as the principal game bird of eastern South Dakota, little information has been available regarding the parasites of the bird in this area. Moreover, few parasite surveys have been reported from other areas of the United States where the ring-necked pheasant has become established in the wild state. The purpose of the present study was to ascertain the incidence of helminth parasites in the pheasants from a single representative county and to investigate any correlation between parasitism and age of bird, seasonal distribution of parasites, and any gross pathology which might be associated with parasitism. Brown County forms part of the northern boundary of eastern South Dakota. Its physiographic position is in the flat James River basin in the glaciated Central Lowlands province of the Mississippi River drainage basin. Food and shelter are plentiful, and characteristically a large pheasant population is supported. We considered this to be a representative locality in which to conduct the study. Acknowledgment is due the South Dakota Department of Game, Fish and Parks for assistance in collecting pheasants. The present report has been taken from a thesis submitted by the senior author in partial fulfillment of the requirements for the M.S. degree.

Viscera from 390 wild turkeys (Meleagris gallopavo) from 31 collection areas in 10 southeastern states, from 76 pen-raised wild turkeys from Beaufort County, South Carolina, and from 68 domestic turkeys from 7 farm areas located in the states of Florida, Virginia, and West Virginia were examined for helminths. Species of helminths and infection percentages of wild turkeys (=W), penraised wild turkeys (=P), and domestic turkeys (=D) follow: TREMATODA-Brachylaema virginiana 4.4 percent (W); Cotylurus flabelliformis 4.4 percent (W); Echinoparyphium recurvatum 7.2 percent (W); Zygocotyle lunata 1.8 percent (W). CESTODA-Davainea meleagridis 11 percent (W); Hymenolepis cantaniana 4.1 percent (W), 1.5 (D); Hymenolepis carioca 10.5 percent (W), 7.9 (P), 1.5 (D); Metroliasthes lucida 43.1 percent (W); Raillietina georgiensis 6.2 percent (W), 18.4 (P); Raillietina ransomi 12.6 percent (W), 6.6 (P), 2.9 (D); Raillietina williamsi 39.0 percent (W), 27.6 (P); Hymenolepid 2.6 percent (W). NEMATODA--Ascaridia dissimilis 66.2 percent (W), 71.1 (P), 35.3 (D); Ascaridia galli 1.5 percent (W), 3.9 (P), 17.6 (D); Capillaria bursata 1.3 percent (W), 3.9 (P); Capillaria obsignata 9.7 percent (W), 73.7 (P), 14.7 (D); Heterakis gallinae 62.3 percent (W), 53.9 (P), 36.8 (D); Strongyloides avium 0.3 percent (W); Strongyloides sp. 0.5 percent (W); Trichostrongylus tenuis 5.1 percent (W), 6.6 (P). The following nematodes were reported present in one or more birds: Capillaria annulata (W); Dispharynx nasuta (W) and (P); Gongylonema ingluvicola (W) and (P); Seurocyrnea colini (W) and (P); Seurocyrnea sp. (W) and (P). The two flukes (Brachylaema virginiana and Cotylurus flabelliformis) collected from wild turkeys constituted new host records. Numerous new state records of turkey helminths were established. The high percentage of Capillaria obsignata in pen-raised birds indicates the danger of introducing these birds to new locations. The high infectivity rates of all groups with Heterakis gallinae should not be overlooked because the ova of the cecal worm transmit the etiological agent (Histomonas meleagridis) of blackhead. Information on the geographic distribution and incidence of gastrointestinal helminth infections of wild and pen-raised wild turkeys is limited. In an effort to correct this deficiency, a survey of parasitism in turkeys was conducted, and the findings were compared with a similar study on domestic turkeys. Gardiner and Wehr (1949), Mosby and Handley (1943), and Self and Bouchard (1950) have published limited reports on the incidence of helminth infections among wild turkeys. The interrelationships of parasitism among wild, pen-reared wild, and domestic birds have not been established: for instance, Reid and Nugara (1961) found substantial evidence that a new cestode species (the tapeworm Raillietina georgiensis) had been introduced into a flock of domestic turkeys by penraised wild birds from Fitzgerald, Georgia. This study was partially financed by a National Science Foundation Grant (No. G-12999), by the Southeastern Association of Game and Fish Commissioners, and by the U. S. Fish and Wildlife Service (Region 4). The present report has been condensed from a thesis submitted by the senior author in partial fulfillment of the M.S. degree. For procuring viscera of wild and penraised wild turkeys, specific appreciation is expressed to biologists of the game and fish agencies of Alabama, Arkansas, Florida, 1 Journal series paper No. 262, College Experiment Station, University of Georgia, Athens. 2 Present address: Director, Poultry Diagnostic Laboratory, Charles Pfizer & Company, Inc., Cullman, Alabama.

Although phenothiazine and piperazine are anthelmintics commonly used in poultry, reports differ as to their value against the common poultry parasites, Heterakis gallinae and Ascaridia galli. Roberts (1940) and Allen, Olivier, and Peterson (1942) indicated that phenothiazine showed anthelmintic action against both Heterakis and Ascaridia; on the other hand, Guilhon (1948), Martin Vaquero (1952), and others found that phenothiazine had little, if any, effect against Ascaridia. Sloan, Kingsbury, and Jolly (1954), Shumard and Eveleth (1955), Shumard (1956), and others found that piperazine exhibited effective action against Ascaridia in poultry. Shumard (1957) found also that piperazine exhibited some anthelmintic action against Heterakis. Edgar, Davis, and Frazier (1957) obtained erratic results against Heterakis with piperazine compounds in water, feed, tablet, and capsule, although they felt that piperazine was about as effective as phenothiazine. Polakova (1957), using piperazine adipate, reported an efficacy of 23 percent against Heterakis. Because of these conflicting reports, trials were undertaken to determine the effectiveness of phenothiazine, piperazine, and mixtures of the two compounds against Heterakis and Ascaridia in chickens.