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The pathological effects of infections of Dispharynx nasuta (Nematoda : spiruroidea) on the blue grouse… Jensen, Doris Nestler 1962

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THE PATHOLOGICAL EFFECTS OF INFECTIONS OF PISPHARYNX  NASUTA (NEMATODA:SPIRUROIDEA) ON THE BLUE GROUSE DENDRAGAPUS OBSCURUS (SAY) by DORIS NESTLER JENSEN B.A., U n i v e r s i t y of Toronto, 1 9 5 5 M.A., U n i v e r s i t y of Toronto, 1 9 5 7 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Zoology We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA August 1 9 6 2 In presenting thxs thesis in p a r t i a l fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It i s understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of ^ ^ s ^ ^ t ^ The University of British Columbia, Vancouver 8, Canada. Date ^s^^a?* ^, GRADUATE STUDIES Field of Study: Zoology Parasitology Advanced Parasitology Biological Methods Histological Technique Marine Field Course Other Studies Introductory Bacteriology Introduction to Viruses Pathogenic Microbiology Poultry Diseases and Hygiene J.R. Adams J.R. Adams Staff P. Ford P.A. Dehnel J.J. Stock J.E. Bismanis C . E. Do lman J.E. Lancaster The University of British Columbia FACULTY OF GRADUATE STUDIES PROGRAMME OF THE FINAL ORAL EXAMINATION FOR THE DEGREE OF -DOCTOR OF PHILOSOPHY of DORIS ELAINE NESTLER JENSEN B.A. (Honours), University of Toronto,: 1955 M.A. , University of Toronto, 1957 FRIDAY, AUGUST 10, 1962, AT 8:30A.M. IN ROOM 3332, BIOLOGICAL SCIENCES BUILDING COMMITTEE IN CHARGE Chairman: F. H. SOWARD J.F. BENDELL C.V. FINNEGAN J. BIELY W.S. HOAR . I. McT. COWAN P.A. LARKIN P.A. DEHNEL J.H. MATHER A. J. WOOD External Fisheries Examiner: L. MARGOLIS Research Board of Canada THE PATHOLOGICAL EFFECTS OF INFECTIONS OF DISPHARYNX.NASUTA (NEMATODA:SPIRUROIDEA) ON THE BLUE GROUSE DENDRAGAPUS OBSCURUS (SAY) ABSTRACT The pathological effects of infections of Dispharynx  nasuta (Nematoda:Spiruroidea) on confined, experiment-ally infected chicks of the blue grouse, Dendragapus  obscurus, have been studied. The severity of'the infection was directly proportional to the number of worms present and the youth of the host. The development of the lesion produced at the site of infection, the proventriculus of the host, is described and its papillomatous nature confirmed. The previously unknown development of stages of D. nasuta in the avian host are described and related both to the formation of the lesion and to the disease process. The growth of infected birds, expressed as gain in weight, was less than that of the controls although food intake studies indicated that the amount of food eaten by both groups was comparable. Calcium and phosphorus analyses on bones gave no indication that the mineral metabolism of infected birds was affected although their bones broke more readily than those of the controls. Development of the juvenai feathers which appear from 3 to 5 weeks of age may be severely retarded. These observations suggest that the protein metabolism of the host is impaired. The numbers of hemocytes of infected grouse fluctuate greatly and show two critical low periods. The first, occurring immediately after infection, can be correlated with the invasion of the larvae and possibly to a substance secreted by them. The second occurs 2 to 3 weeks after i n i t i a l infection and can be correlated with local irritation and hemorrhages and perhaps the moult of the larvae. Chronic hematological symptoms are anemia and leucocytosis. The latter is characterized by he.terophilia, eosinophilopenia, lymphocytosis of small forms and lymphopenia of the larger forms. The presence of circulating- antibodies for D. nasuta was not demonstrated with the techniques used. The evidence suggests that the host tissue reaction may be an allergic response. Several ecological questions concerning the survival of D. nasuta during the winter months, temperature for larval development in the inter-mediate host, longevity of adult D. nasuta in the definitive host, infection and reinfection of adult blue grouse, are considered. In the laboratory,, infections of 16 to 22 worms administered to the host before 2 weeks of age were fatal. Results of these experiments indicate that D. nasuta is a debilitating pathogen which may prove fatal and may act as a controlling factor of natural grouse populations. Abstract The pathological effects of infections of Dlspharynx  nasuta (Nematoda: Acuariidae) on confined, experiment ally-infected chicks of the blue grouse, Dendragapus obscurus, have been studied. The severity of the infection was found to be directly proportional to the number of worms present and the youth pf the host. The development of the lesion produced at the site of infection, the proventriculus of the host, is described and i t s papillomatous nature confirmed. The previously unknown develop-mental stages of D. nasuta in the avian host are described and related both to the formation of the lesion and to the disease process. The growth of infected birds, expressed as gain in weight, was less than that of the controls although food intake studies indicated that the amount of food eaten by both groups was com-parable. Calcium and phosphorus analyses on bones gave no in-dication that the mineral metabolism of infected birds was affected although their bones broke more readily than those of the controls. Development of the Juvenal feathers which ajpear from 3 to 5 weeks of age may be severely retarded. These obser-vations suggest that the protein metabolism of the host is impaired. The numbers of hemocytes of infected grouse fluctuate greatly and show two c r i t i c a l low periods. The f i r s t , occurring immed-iately after infection, can be correlated with the invasion of the larvae and possibly to a substance secreted by them. The second occurs 2 to 3 weeks after i n i t i a l infection and can be correl-ated with local i r r i t a t i o n and hemorrhages and perhaps the moult of the larvae. Chronic hematological symptoms are anemia and leucocytosis. The latter is characterized by heterophil ia» eosinophilopenia, lymphocytosis of small forms and lymphopenia of the larger forms. The presence of circulating antibodies for D. nasuta was not demonstrated with the techniques used. The evidence suggests that the host tissue reaction may be an allergic response. Several ecological questions concerning the survival of D. nasuta during the winter months, temperature for larval devel-opment in the intermediate host, longevity of adult D. nasuta in the definitive host, infection and reinfection of adult blue grouse, are considered. In the laboratory, infections of 16 and 22 worms, administered to the host before 2 weeks of age, were f a t a l . Results of these experiments indicate that D. nasuta is a debilitating pathogen which may prove fatal and, may act as a controlling factor of natural grouse populations. A C K N O W L E D G E M E N T S Throughout the course of the study I have received help and encouragement from many sources. To a l l those who have participated in any way I extend my most sincere gratitude. In particular, I wish to express my appreciation to Dr. J. R. Adams, director of the study, for stimulating counsel, encouragement, and for suggestion of the problem; to Dr. J. P. Bendell, Professor J. Biely (Dept. of Poultry Science), Dr. P. A. Dehnel, Dr. H. E. Taylor (Dept. of Pathology), members of the committee, for helpful suggestions jn particular phases of the work, and for c r i t i c a l l y reviewing the manuscript; to Dr. I. MoT. Cowan, chairman of the committee, for c r i t i c a l l y reviewing the manuscript and for the provision of laboratory f a c i l i t i e s ; to Mr. George Gibson for assistance and helpful criticism in a l l phases of the work; to Miss Mia van Eerten, M J S S Sonia Kindrachuk and Mr. Edward Platzer for technical assistance; to Dr. N . J. Wilimovsky and Mr. H. Dempster (Computing Centre) for helpful advice with the statistics and computer programming; and to officers of the Provincial Pish and Game Department for their cooperation in the f i e l d . . The financial assistance of the NationalR esearch Council and Canadian Industries Limited is gratefully acknowledged. TABLE OP CONTENTS Page GENERAL INTRODUCTION 1 GENERAL MATERIALS AND METHODS 9 A. Experimental animals 9 B. Growth and development of blue grouse 11 C. Hematology of blue grouse 13 D. Hi s t o l o g y of the p r o v e n t r i c u l u s 15 E. Immunology 15 S e c t i o n I . BIOLOGY OP THE PARASITE 18 Int r o d u c t i o n 18 M a t e r i a l s and Methods 20 Results and Observations 20 Discussion 24-S e c t i o n I I . THE PRO VENTRICULAR LESION 25 The Normal P r o v e n t r i c u l u s 25 The In f e c t e d P r o v e n t r i c u l u s 27 D i s c u s s i o n 4-0 S e c t i o n I I I . EFFECT OP THE PARASITE ON THE HEMATOLOGY OF THE HOST 44 I n t r o d u c t i o n 44-M a t e r i a l s and Methods 44 Hematology of Co n t r o l B i r d s 45 Hematology of In f e c t e d B i r d s 48 D i s c u s s i o n - 54-S e c t i o n IV. EFFECT OF THE PARASITE ON THE GROWTH AND DEVELOPMENT OF THE HOST 62 I n t r o d u c t i o n 62 Page M a t e r i a l s and Methods 63 Growth Rate of C o n t r o l Blue Grouse 66 Growth of I n f e c t e d Grouse 68 Food Intake of Grouse 68 Bone Development of Grouse 68 Feather Development of Blue Grouse 68 D i s c u s s i o n 73 S e c t i o n V . IMMUNOLOGY AND RESISTANCE 76 Immunology 76 R e i n f e c t i o n Test 77 Age R e s i s t a n c e 77 D i s c u s s i o n 79 S e c t i o n VI. APPEARANCE, BEHAVIOUR AND SURVIVAL OF INFECTED HOSTS 83 Appearance and Behaviour 83 S u r v i v a l 84 GENERAL DISCUSSION 85 SUMMARY AND CONCLUSIONS 97 LITERATURE CITED 102 APPENDIX A. Determination of Age of Chicks Caught i n F i e l d 107 APPENDIX B. A n a l y s i s of Lo g a r i t h m i c Transformation of Growth Data 110 APPENDIX C. Experimental I n f e c t i o n s of the Domestic Fowl w i t h D. nasuta I l l i i i List of Tables Page I Endoparasites reported from blue grouse on Vancouver Island 7 II Developmental stages of D. nasuta found in the prov-entriculus of experimentally infected blue grouse correlated to the age of the infection 2 2 III Means and ranges of values for erythrocytes, polychrom-atic erythrocytes, leucocytes and hemoglobin obtained for control blue grouse 50 IV Means and ranges for control blue grouse, of values for various leucocytes per cu. mm. of blood 5 0 V Values for blue grouse chicks of "m" and "b" in the linear growth equation y = mx + b using the Walford transformation of weight data 66 VI Relationship between quantity of food consumed and gain in weight of infected and control blue grouse 7 1 VII Relationship between calcium and phosphorus in femurs of infected and control grouse chicks 7 2 VIII Results of incubation of 3rd stage larvae of D. nasuta in a variety of media 78 IX Computed growth rates for blue grouse of two sexes and two subspecies raised in captivity 1 1 0 iv List of Figures Facing page Fig. 1. Esophagus, proventriculus, interventriculus and gizzard of infected and normal blue grouse..35 Fig. 2. Mucosal surface of normal proventriculus 35 Fig. 3. Infected proventriculus, entire 35 Fig. 4. Infected and normal proventriculi opened to show mucosal surface 35 Fig. 5 . Mucosal surface of infected proventriculi en-larged to show worms in situ 35 Fig. 6. Plicae and sulci of the mucosal epithelium of the normal proventriculus 36 Fig. 7. Mucous glands and squamous epithelium of the esophagus 36 Fig. 8. Section through middle portion of the normal proventriculus showing arrangement and relative thicknesses of the tissues 36 Fig. 9. Arrangement of the apocrine cells of the deep glands and the muscular tunics ' 36 Fig. 10 . Mucosal epithelium of the proventriculus one week after infection showing worm in situ 36 Fig. 11. Longitudinal sections of whole proventriculi taken 1, 2 and 8 weeks after infection 37 Fig. 12. Longitudinal section of whole proventriculus taken 12 weeks after infection 37 Fig. 13. Enlargement of plicae of mucosal epithelium 1 week after infection 37 V Pacing page Fig. 3.4-. Cellular exudate in plicae 1 week after infection 37 Fig. 15. Mucosal epithelium 2 weeks after infection 38 Pig. 16. Cellular exudate in plicae 2 weeks after infection .38 Fig. 17. Mucosal epithelium 3 weeks after infection 38 Fig. 18. Cellular exudate in plicae 3 weeks after infection 39 Fig. 19. Mucosal epithelium 4- weeks after infection 39 Fig. 20. Papilloma of the squamous epithelium of the esophagus 12 weeks after infection 39 Fig. 21. Portion of the mucosal epithelium 12 weeks after infection 39 Fig. 22. Hemocytes of the blue grouse 49 Fig. 23. Relationship between the numbers of certain hemocytes present in the blood of blue grouse and the time elapsed after infection. A. erythro-cytes, B. polychromatic erythrocytes, and C. leucocytes 51 Fig. 24-. Relationship between the numbers of certain hemocytes present in the blood of infected blue grouse and the time elapsed after infection. A. total lymphocytes, B. small lymphocytes, and C. large lymphocytes 52 vi Facing page Fig. 25. Relationship between the numbers of certain hemocytes present in the blood of infected, blue grouse and the time elapsed after i n -fection. A. heterophils, B. eosinophils and C. monocytes 53 Fig. 26. Walford transformation of weight data from 2 sexes and 2 subspecies of blue grouse chicks 65 Fig. 27. Relationship between weight gain of infected blue grouse chicks and (a) the age at which the infection was acquired (b) the number of worms present 67 Fig. 28. Relationship between the growth of 19 control ruffed grouse and a single ruffed grouse infected with 11 D. nasuta 70 GENERAL INTRODUCTION The nematodes have been implicated more frequently as pathogens than the other types of parasitic helminths. Not a l l nematodes are pathogenic. Some may cause no detectable change in the host when present in small numbers and are considered benign. However, larger numbers of the same parasite may be ex-tremely damaging to the host and may even prove fata l (e.g., Amidostomum anseris, Capillaria annulata, Syngamus trachea). Other nematodes may cause detectable damage even when they occur in small numbers. In such cases, there is usually extensive host reaction to the invading organism. The types of damage effected by parasitic nematodes are diverse and may be classif i e d as mechanical, biochemical and de-privational. Mechanical effects include, (a) obstruction or occlusion of organs or vessels in which the parasite is situated (e.g., Synagamus sp. in the trachea of birds, Wuchereria bancrofti in the lymphatic vessels of man); (b) i r r i t a t i o n producing local chronic inflammation, one result of which i s tissue proliferation (e.g., Gongylonema neoplasticum in the stomach of rats); (c) des-truction of epithelial integrity which may provide portals of entry for secondary bacterial invasion; (d) introduction by the nematode of a pathogen (e.g., Heterakis galllnae may act as a vector for Hlstomonas meleagrldls the causal agent of blackhead in the turkey). The biochemical effects of nematode infections are less well known. Histolytic substances are produced by migrating larvae - 2 -(e.g., Asoarls lumbriooides) and anti-coagulants by nematodes which feed, on the blood of the host (e.g., Ancylostoma canlnum). The possible elaboration by nematodes (e.g., Asoarls lumbricoides) of anti-enzymes which inhibit the action of trypsin and. pepsin may reduce protein digestion in affected hosts. Nematode parasites may compete with the host for essential nutrients or may deprive the host of materials which i t has elaborated. The anemias resulting from infections of blood feed-ing nematodes (e.g., Hemonchus sp., Necator sp. and Ancylostoma spp.) are particularly well documented. Dlspharynx nasuta is an example of a nematode parasite which produces spectacular changes in i t s host even when present in small numbers. The presence of even one D. nasuta w i l l consist-ently e l i c i t a predictable, extensive host reaction in the form of a lesion in the proventriculus at the site of infection. It has been reported frequently as the causative agent of fatal disease in domestic fowl (Legros, 1864; Allcata, 1951 and many others), in pigeons (Cram, 1928) , in ruffed grouse (Edminster, 19^7) and in the catbird. (Cram, 1932) . Allen (1925) believed, that D. nasuta was the chief cause of "Grouse Disease" in the northeastern United States. However, he later altered this view when i t was found that the distribution of D. nasuta was discon-tinuous, and. concluded that this nematode was a serious path ogen wherever i t occurred, but that other unknown factors must be res-ponsible for the loss of grouse where D. nasuta was absent (Allen and Gross, 1926) . Several investigators of the fluctuations in ruffed grouse populations have tried, unsuccessfully, to correlate - 3 -D. nasuta infections with major population declines (Allen, 1925; Edminster, 194-7). The latter author stated that, "infections with 20 to 30 worms are sufficient to affect the "bird's health and those more severe may prove f a t a l " . In 1955, Bendell conducted a study of the population dynamics of the blue grouse Dendragapus obscurus fuliginosus (Ridgway) on Vancouver Island. He concluded that D. nasuta in conjunction with the acanthocephalan, Plagiorhynchus formosus Van Cleave, were causal agents of the heavy mortality which he recorded for blue grouse chicks. This mortality was calculated to be a 67$ loss of chicks per hen on the summer range. As well, he suggested that these parasites were carried by the birds into the winter period and that "chicks carrying these parasites would suffer greater mortality under winter stress than would the older age classes (chick) mortality was calculated at 4-7$ between f a l l and spring as compared to a yi$ yearly mortality in the older birds." His studies also indicated that only 4$ of yearling birds were infected with D. nasuta (from 1 to 6 worms) and that adult birds were never infected. This i s a very low incidence of i n -fection when compared with the 64$ infection (from 1 to 4-30 worms) he found in chicks from time of hatch to August of the same year. Although D. nasuta is considered an important pathogen and, indeed, as a controlling factor in grouse populations, no work has been done on the disease process or mortality rate apart from studies on the tissue changes in the proventriculus and descrip-tions of the signs of the disease. - 4 -The proventricular lesion has attracted a considerable amount of attention and conflicting interpretations of its structure exist in the literature. It was f i r s t described in detail by Kasielewski and Wiilker in 1919 from a single German carrier pigeon. They described the papillomatous nature of the tumour-like growth associated with the nematode but were unable to study the development of the lesion. Cram (1928) described the lesion from a pigeon as, macroscopically, a "thick necrotic mass" and noted that microscopically, "there appeared to be a l -most complete destruction of the glands and a marked cellular Infi l t r a t i o n of the underlying tissue". Wehr (1948) attributed the formation of ulcers in the proventriculus to this nematode. He stated that, "In case of heavy infestations, the wall of the proventriculus becomes tremendously thickened and macerated, tissue layers are indistinguishable, and the parasites become a l -most completely concealed beneath the proliferating tissue." Levine and Goble (1947) mentioned severe inflammation of the pro-ventriculus of the ruffed grouse (Bonasa umbellus) with thickening of the proventricular wall, destruction of the glandular tissue and the muscle layers. They also noted that destruction leading to perforation of the glandular stomach may cause fatal peritonitis. Tissue necrosis, destruction of the muscularis, and. ultimate t e l -escoping of the entire proventriculus into the gizzard were also described. More recently, Hwang, Tolgay, Shalkop and Jaquette (1961) published a histopathological description of the tissue change produced in the proventriculus of a pigeon by D. nasuta. In - 5 -summary, they described the lesion as a catarrhal type of i n -flammation characterized by epithelial desquamation, papillary proliferation, hypersecretion of mucous, congestion and secondary bacterial invasion of the superficial mucosa. As well as the tissue changes in the proventriculus of the affected host, certain disease signs have been attributed to JD. nasuta infections. In 1864-, Legros reported an epizootic caused by this nematode in domestic fowls in France. Affected birds appeared dejected, became emaciated without loss of appetite and died from apparent exhaustion. Similar signs were described by Cram (1928) for a carrier pigeon which died of D. nasuta infection in North America. In addition, she reported that loss of pigment from the iris,"probably due to anemia produced by the worms", was a "marked feature associated with the disease". Bump (1947) des-cribed emaciation and sluggish flight of infected ruffed grouse. There seems to be no doubt that D. nasuta is a pathogen. Ex-perimental proof, however, that these infections can prove fatal in the absence of other parasites and adverse environmental con-ditions is lacking. Nor is there any information concerning the course of the disease, the significance of numbers of the nema-todes, the specific effects of the infection on the host or how these effects are produced by the parasite. To supply this information, i t was necessary to obtain parasite and disease free hosts and maintain them in this con-dition. This necessitated a laboratory approach to the problem. The opportunity of studying parasite free hosts in the f i e l d , or hosts infected by p. nasuta alone is non-existent due to the - 6 -variety of endoparasites to which D. obscurus is normally a host. A l i s t of the endoparasites reported from blue grouse on Vancouver Island is presented in Table I. Another advantage of the laboratory approach to the problem was that controlled dosages of larvae could be administered to chicks of various ages. In this way, the effects of numbers of D. nasuta and the relationship between severity of the reaction and maturity of the host could be determined. Since there are, in the literature, conflicting interpret-ations of the nature of the proventricular lesion i t was necessary to trace the changes in the affected tissues from the i n i t i a l stages of infection and to ascertain the relationship of D. nasuta to these tissues. Only by this method could the complicated structure of the advanced lesion be clearly defined. The various signs of D. nasuta infections reported in the literature suggested particular aspects of the disease which might be pursued under controlled conditions. Cachexia without anorexia (Cram, 1928; Legros, 1864) suggested studies on the growth and food intake of infected birds. Apparent anemia (Cram, 1928) sug-gested a study of the hematology of infected birds and also a search for occult blood in the faeces of these birds. Bone dev-elopment and feather production were studied when i t became evident that these processes were also affected by the disease. Although the reports of D. nasuta infection are not always clear as to the age class infected, there is no lack of evidence that adult birds are frequently infected (Gross, 1925; Edminster, 1947). Consequently, the low incidence of D. nasuta infections Table I Endoparasites reported from blue grouse on Vancouver Island Parasite Site Investigator Leucocytozoon sp. Protozoa Haemoprqteus sp. " Trypanosoma sp. " Dispharynx nasuta Nematoda Chellospirura spinosa " Yseria sp. " Ascaridla bonasae " Splendidofilaria sp. " Rhabdometra n u l l l o o l l i s Cestoda Braohylalma sp. Trematoda Blood Bendell, 1955 Proventriculus Gizzard II Intestine nr. blood Gibson (personal vessels communication) Intestine Bendell, 1955 Jensen (this study) Plaglorhynchus formosus Acanthocephala Bendell, 1955 - 8 -in yearling blue grouse and the non-discovery of such infections in adult birds by Bendell (1955) posed interesting questions. To answer them, studies were conducted on the longevity of !?• nasuta and on the susceptibility to infection and reinfection of yearling and adult birds. A search was also made for evidence of circulating antibodies for D. nasuta larvae and adults. An attempt was made to use chicks of domestic fowl as exper-imental animals during the winter months when blue grouse chicks were not available. The results of these t r i a l infections are also presented. - 9 -GENERAL MATERIALS AND METHODS A. Experimental animals (i) Source and maintenance of definitive hosts Blue grouse of the two subspecies, Dendragapus obscurus  fuliginosus (Ridgway) and D. o. pallldus Swarth were utilized as experimental hosts. Chicks of D. o. fuliglnosus were collected on Vancouver Island at ages ranging from 3 to 20 days. D. _o. pallldus were hatched from partially incubated clutches collected in the Okanagan Valley. The eggs were transfered in thermos jugs containing warm grain to Vancouver where incubation was completed in a Jamesway forced a i r incubator at 37-5° c -The chicks were brooded in a commercial electric battery brooder and fed Turkey Starter Crumbles containing 28$ protein (Buckerfields Ltd.). At 6 weeks of age they were moved to i n -dividual wire mesh cages and maintained in a flyproof poultry house. At 8 weeks of age, the diet of the chicks was gradually changed to Chicken Grower Pellets containing 1$% protein (Bucker-fields Ltd.). Water and granite grit were available at a l l times and the diet was regularly supplemented with green leafy veget-ables. Terramycin (Phizer, Poultry Formula) was added to the drinking water at a preventative level as a safeguard against bacterial infections. Two partially incubated clutches of ruffed grouse eggs, Bonasa umbellus (L.) were obtained from Vancouver Island. These and several day-old domestic chicks from the poultry farm at the University of Br i t i s h Columbia were maintained in the same manner as the blue grouse eggs and chicks. - 10 -A limited number of blue grouse chicks were obtained by breeding adult blue grouse within the colony of experimental birds. ( i i ) Source and maintenance of intermediate host species. Sow bugs of the two locally common species Poroellio  scaber L a t r e i l l e , and Oniscus asellus L. were used as intermediate hosts. These were collected from compost from local gardens and were maintained in the laboratory on moist s o i l or peat moss in glass containers. Lettuce or carrots were occasionally supplied as food. ( i i i ) Source and culture of Dispharynx nasuta Adults of D. nasuta were recovered in September from infected proventriculi of blue and ruffed grouse passing through the game checking stations on Vancouver Island. In the laboratory, the female worms were dissected and the eggs spread on discs of carrot. Several treated carrot discs were placed on moist f i l t e r paper in a 4 inch stacking dish with 25 sow bugs. The dishes were kept moist and maintained at 2 2 °C. for at least 35 days to allow development of the D. nasuta larvae to proceed to the third stage. Infected sow bugs were stored in a cool room at 1 1 . 5 ° C until used. Attempts were made to use the faeces of infected birds as a source of D. nasuta eggs. However, concentration techniques failed to recover large numbers of the eggs and faeces rapidly deteriorated releasing gases which ki l l e d the sow bugs to which they were fed. - 1 1 -(iv) Procedure used in infecting the definitive hosts. Infected sow bugs were dissected in 0.85$ saline. Third stage larvae of D. nasuta were recovered and transferred to fresh saline. Suitable infective doses were collected in a pipette and given to the definitive host by mouth. No attempt was made to starve the chicks before administering the larvae. The infection was verified by examination of fecal smears. The presence of eggs of D. nasuta in the smears 27 to 30 days after the dosage indicated the successful establishment of the larvae. B. Growth and development of blue grouse. (i) Pood intake of Infected and control chicks. During one phase of the experiment, the food intake of in -fected and control birds was recorded. Each chick was caged sep-arately and fed from individual food dishes. The weight, to the nearest gram, of food removed from the dishes over each 24- hour period was recorded. Pood dishes were never allowed to empty and spilled food was weighed so that an accurate measure of the food consumed could be obtained. ( i i ) Weight increase in grouse chicks. Increase of weight of chicks was used as the criterion for growth. Attempts were made to correlate weight with the com-bined lengths of the tibiotarsus and the tarsometatarsus. This last measurement proved d i f f i c u l t to obtain and involved excessive handling of the bird. It was, therefore, discontinued. A series of control chicks was weighed'daily until 8 weeks - 12 -of age. Prom 8 to 20 weeks, weights were recorded twice a week. A second, series of control and infected birds were weighed, on alternate days until 8 weeks of age and weekly un t i l the period of experimentation terminated at 15 weeks. Birds were restrained in a cotton or paper bag of known weights and weighed to the nearest 0,1 g. Growth rates were determined for male and female controls of each subspecies, and the growth of infected birds compared with these values. Similar data were obtained for a series of ruffed grouse chicks. However, in this species, differences in growth rate due to sex could not be assessed because of the d i f f i c u l t y m sexing the birds. ( i i i ) Feather growth of blue grouse chicks. The feather development of control chicks of known age was used to determine the age of chicks caught in the f i e l d . This development was expressed 3 ways, (a) as the length of feathers measured with a ruler to the nearest millimeter, (b) as the length of feathers expressed relative to points on the body (i.e. age noted when primaries of folded wing reached the base of the pygidium), (c) as the age at which feathers in the various tracts appeared as tufts at the distal tips of the peridermal sheaths of the feather papillae. Comparisons were also made between the rate of development of feathers in infected and control birds. (iv) Chemical analysis of bones of blue grouse. The femur was selected for analysis for 2 reasons: (a) i t is a large pneumatic bone containing no marrow, (b) i t was frequently bowed or broken in infected birds. - 13 -One femur was removed from the body of each of several infected and control birds, freed of muscles and tendons and dried to constant weight (determined on a Sartorius electric balance to the nearest 0.0001 g.). Subsequent to muffling and ashing, the bone samples were allowed to cool and were reweighed to the same order of accuracy to obtain the ratio of dry weight to ash weight. Phosphorus determinations were performed by reduction of phosphomolybdic acid by ferrous sulphate (Taussky and Shor, 1953) . Calcium levels were determined by precipitation of calcium as the oxalate which was titrated in an acid medium (H2S0^) against standardized K Mn 0^ solution (Kramer and T i s d a l l , 1921) , (Clarke and Collip, 1925) . C. Hematology of blue grouse (i) Sampling technique Blood was obtained by needle puncture of the brachial vein. Three drops of blood were allowed to flow freely and samples for examination were drawn immediately the blood appeared. If, as occasionally happened an insufficient quantity of blood were ob-tained, (due to rapid coagulation) the bird was returned to i t s cage and resampled 24- hours later from the opposite vein. Every effort was made to disturb the birds as l i t t l e as possible during sampling. ( i i ) Determination of Hemoglobin level The standard technique using a Sahli hemometer was used, as the acid hematin formed from bird blood varies only slightly - 14 -in hue from the colour standards. ( i i i ) Hemocyte counts. The blood in a standard Thoma red blood c e l l pipette was diluted 1:200 with a solution of sodium citrate in 0.85$ saline. Pipettes were shaken by hand and 2 c e l l counts made on a Spencer Bright Line hemocytometer chamber within 15 minutes of sampling. If the difference between the 2 counts was greater than 10, the results were discarded and a new sample taken from the pipette. Three blood smears were made at each sampling. These smears were fixed in absolute methanol and stained with Wright's blood stain. They were subsequently used (a) to determine the ratio of polychromatic erythrocytes to mature erythrocytes, (b) to determine the ratio between erythrocytes and leucocytes, (c) to differentiate the various types of leucocytes. The method used was the indirect technique for counting avian blood cells des-cribed by Schmeisser (1916) . (iv) Differentiation of leucocytes. Differential white blood c e l l counts were made on the stained smears under o i l immersion using the four-field meander method (Holman, 195&). One thousand leucocytes were counted and differentiated for each sample. Identification of the various leucocytes was based on Pantham ( 1910) , Porkner (1929) and Olsen (1948) . The following categories were used: (a) heterophils orpseudoeosinophlls (b) eosinophils (c) basophils (d) monocytes (e) large lymphocytes (f) small lymphocytes (g) unidentifiable or extremely young cells - 15 -of doubtful identity. The validity of these categories was confirmed by the recent work of Lucas and Jamroz ( I 9 6 I ) on the blood of the domestic fowl. (v) Test for occult blood in the feces. The benzidine test (from Boddie, 195&) was used to det-ermine the presence of blood in feces. Fresh feces from both control and infected birds were tested. D. Histology of the proventriculus. (i) Source of tissues Proventriculi, including the posterior portion of the esophagus and the anterior region of the interventriculus, were collected from experimentally and naturally infected blue grouse. Normal proventriculi were collected from laboratory control birds only. ( i i ) Histological procedures A l l tissues were fixed in 10$ formalin, embedded in paraffin, and sectioned longitudinally at 10 microns. Routine serial sections were stained with Heidenhain's haematoxylin and eosin (Lee, 1950) . Several sections were stained with Hale's stain and the Periodic acid-Schiff (P.A.S.) techniques* by. the Pathology Department of the University of Brit i s h Columbia. E. Immunology (i) Preparation of antigen from adult worms An aqueous extract of powdered, dessicated adult D. nasuta - 16 -was prepared after the method described by Markell and Voge (1958). Two preparations were made, one from 150 fresh males and one from l 6 l frozen female worms. ( i i ) Preparation of antigen from third stage larvae The small size and the d i f f i c u l t y of obtaining large numbers of third stage larvae eliminated the possibility of a larval extract. On the hypothesis that secretions or metabolic products of the larvae would have antigenic properties, twenty larvae were incubated at 39° C. for 3 days in 5 cc. of 0.85$ saline. The saline was collected, f i l t e r e d and refrigerated 2 weeks un t i l used. ( i i i ) Collection of plasma from birds Blood was collected from the brachial vein of infected birds using a sterile syringe and a No. 27 hypodermic needle. From 3 to 5 c c ' were recovered at a time. The blood was dis-charged into sterile tubes and centrifuged u n t i l the supernatant was clear. The plasma was pipetted off and refrigerated 2 weeks un t i l used. (iv) Test for perilarval precipitates Third stage larvae of D. nasuta were incubated at 39° C. in sterile well slides containing plasma from infected birds. Saline and plasma from non-infected birds were used as controls. (v) In vitro test for precipitation of antigen-antibody complex A modified Ouchterlony (1948)gel-diffusion technique was - 1 7 -used. Petri plates of saline agar were prepared. Three c i r -cular wells 2 cm. apart and 1 cm. in diameter were cut in the agar. The center well was f i l l e d with plasma from infected or control birds and outside wells received larval antigen in the le f t and adult antigen in the right. The plates were kept at room temperature and examined frequently for 2 weeks. (vi) Intradermal test The skin of birds i s exceedingly thin and a suitable site for intradermal injection is d i f f i c u l t to find. The lip s of feather f o l l i c l e s seemed to offer the thickest skin and injec-tions were made in these sites. Larval and adult antigens (approximate dose 0.025 cc. of each) were injected in different areas. Uninfected birds were used as controls for comparison with infected birds and those that had recovered from an infec-tion. ( v i i ) Reinfection experiments Birds which had recovered from their infections by natural means (determined by the lack of p. nasuta eggs in the faeces) were given doses of third stage larvae. - 18 -Section I BIOLOGY OP THE PARASITE Introduction Dispharynx nasuta, a nematode of the Superfamily Spirur-oidea, was f i r s t described by Rudolphi in 1819 from the proven-triculus of the house sparrow Passer domesticus (L.) in Vienna. Mature females of this nematode are approximately 10 mm. in length and relatively broad. Male specimens are approximately 8 mm. in length, slender, and have a spirally coiled t a i l . Both sexes are equipped with four wavy cuticular cordons which orig-inate at the li p s and pass backwards in the cervical region. The dist a l ends of these cordons are recurrent but do not anastomose. p. nasuta has been reported from the proventriculus of many species of galliform, columbiform and passeriform birds (Goble and Kutz, 1945). It seems to be ubiquitously distributed where-ever the ranges of suitable intermediate and definitive hosts coincide. The genus Dispharynx was synonymized with the genus Synhimantus by Osche (1955) , reinstated by Sobolev (1957) and reduced to subgeneric status by Chabaud and Petter ( 1 9 5 9 K The classification of the last mentioned authors Is arbitrary and inconsistent in certain respects and for the present I prefer to consider the genus Dispharynx as distinct and separate from Synhimantus. In I897, Pianna suggested, from studies on related nematodes, that the l i f e cycle of Dispharynx probably involved a terrestrial crustacean as an intermediate host. This was found to be correct - 19 -by Cram (1931) who established, experimentally, the following l i f e cycle for Dispharynx spiralis (synonym of D. nasuta; Goble and Kutz, 194-5). Embryonated eggs laid by the adult female nematodes in the proventriculus of the host were voided with the faeces. If the eggs were eaten by a sow bug, Porcellio  scaber Latr. or Armadillldium vulgare L., they hatched releas-ing f i r s t stage larvae which burrowed through the gut wall of the crustacean into the body cavity. Here the larvae grew and developed. After approximately 14- days, they moulted to form second stage larvae. Third stage larvae were present 26 days after ingestion of the eggs and were then infective to the defin-itive host. Female D. nasuta, gravid with embryonated eggs, were present in the proventriculus of birds 27 days after inges-tion of infected sowbugs. A third intermediate host, Porcellio laevis, was reported for p. nasuta by Edminster (194-7). Several important points, necessary for a f u l l understanding of the development of the lesion and the disease process, are missing from this account. The most important point is whether the larvae penetrate and mature in the tissues of the host or remain in the lumen of the proventriculus. For example, the larval development of Oesophagostomum sp. is completed in the intestine of the host. The larvae of Ascaris lumbricoldes. on the other hand, migrate through the intestinal walls, reach the lungs where they undergo further development, are coughed up and swallowed to become adult in the intestine. In the latter case, pathological effects can be directly attributed to tissue break-- 20 -down and congestion caused by the larval migration. Also those nematodes which migrate come into intimate contact with the tissues of the host and generally e l i c i t strong antibody formation. Furthermore, to understand the significance of the low incidence of D. nasuta infections in yearling birds, and the absence of these infections in adult birds, a knowledge of the a b i l i t y of the worms to survive in their hosts is required. Materials and Methods The times and site at which the larval moults occurred were ascertained experimentally to determine i f any of the observed pathological effects could be correlated to these phenomena. To accomplish this, 5 chicks were each given 12 larvae by mouth and were autopsied at intervals of 3, 5, 7, 10 and 14 days thereafter. Results and Observations (i) Development of D. nasuta in the definitive host The results of the experiments are recorded in Table I I . The larvae found in the proventriculi were identified on the basis of the following descriptions. Redescription of 3rd stage larva of D. nasuta. The description of the 3rd stage larva from sowbugs as given by Cram (1931) is confirmed except for her statement that there was no evidence of cordons. There i s , in the buccal region of this larva, a yellowish substance of a glandular nature which tends to obscure the cuti-cular structures. After invasion of the definitive host, this - 21 -material disappears. Incubation of the larvae in 0.85$ saline at 3 7 ° C for 3 days also results in the disappearance of this substance. Subsequently, 4- delicate cuticular cordons can be distinguished. These cordons, which are entirely straight, originate at the lips and pass backwards for a distance of 72 to 74- microns. Description of the 4-th stage larva of D. nasuta. The body of the 4-th stage larva is 3 .2 to 3 .5 nun. long and 200 to 275 microns wide. The head i s blunt and supplied with a pair of lips from which originate 2 pairs of sinuous cuticular cordons. These cordons extend backwards to a level 197 to 24-4 microns from the anterior end. They are slightly recurrent on their ends and form the shape of a "J". A b i f i d deirid is present 24-6 to 267 microns from the anterior end. The female larvae exhibit a vulva in the posterior third of the body. However, this structure does not appear to be patent. On the t a i l of the male larvae (which i s not alate) two pairs of preanal and two pairs of postanal papillae could be discerned. - 22 -Table II. Developmental stages of D. nasuta found in the proventriculus of experimentally infected blue grouse correlated to the age of the infection. Bird Days after Number of Stage and sex of nemas recov-infection nemas found ered 1 3 4 2 5 11 3 7 1 4 10 10 5 14 8 3rd stage (sex not determinable) 3rd stage- 2m., 2 f. (1 moult-ing); 4 t h stage- 3m., 4 f. 4 t h stage- 1 f. 4 t h stage- 7 f., 1 m. (moult-ing) Immature adults - 2 m . Immature adults - 5 f., 3 m. ( i i ) Ecological relationships between D. nasuta and i t s inter-mediate and definitive hosts. Intermediate hosts of D. nasuta. Both Porcellio scaber Latr. and Oniscus asellus L. were used as experimental hosts. The latter represents a new intermediate host record. 0. asellus proved to be a convenient host animal, supporting larger infections and having a lower mortality rate than P. scaber under laboratory conditions. Effects of temperature on development of D. nasuta larvae. In infected sow bugs held for 35 days at a temperature between 10.5° C and 11.5° C, larval development did not proceed beyond the f i r s t stage. Elevation of the holding temperature to 20° C to 24° C permitted larval development to proceed through the 2nd and 3rd stages. - 23 -Survival of p. nasuta during winter. Sow bugs containing infective 3rd stage larvae have been maintained at 6° C and between 10.5 and 11.5° G for 9 months in the laboratory. There-fore, i t may be possible for D. nasuta to pass the winter as larvae in the intermediate host. Eggs of D. nasuta frozen at -23° C and maintained at this temperature for 18 days remained viable and were infective to sow bugs. Freezing, then, does not k i l l the eggs. This indic-ates that i t may also be possible for this nematode to overwin-ter as eggs in grouse faeces. However, further experimentation to determine the effect on the eggs of prolonged freezing and thawing would have to be performed to prove this point. Some D. nasuta overwinter as adults but the low incidence of infection in yearling birds (4$) and the absence of infec-tions in adult birds would seem to be an inadequate source of infective material to assure transmission of the parasite. Longevity of adult D. nasuta. Examination of the faeces of infected blue grouse revealed the presence of D. nasuta ova up to 9 months after i n i t i a l infection. Subsequent fecal sampling proved negative for ova. Infected birds, on autopsy, have been found to harbour these nematodes up to 11 months after administration of larvae. Female specimens recovered from the host 9 to 11 months after infection appeared spent (i.e. the uterine coils were almost devoid of developing ova and the nematodes appeared to have diminished in length and breadth). A single female nematode was recovered from a bird which had been infected 29 months earlier. Probably the longevity of this worm was abnormally prolonged. - 24 -A healing lesion was found, in the proventriculus of a bird autopsied 14 months after i n i t i a l infection. No p. nasuta were present. Discussion From Table II i t can be seen that both male and female 3rd stage larvae moult to form 4th stage larvae on the 5th day of the infection. Male 4th stage larvae moult to form adults on the tenth day of the infection. Female 4th stage larvae moult later between the 10th and 14th days. It is evident from these results that D. nasuta completes i t s development in the proventriculus of the host. There i s no evidence of larval migration to other host tissues. - 25 -Section II THE PROVENTRICULAR LESION Before the pathological changes in the proventriculus due to infection with D. nasuta could he assessed, i t was necessary to establish the normal morphology and histology of this organ for the blue grouse. Comparison of the morphology of infected and normal proventriculi were based on the examination of this organ in several hundred birds. The normal histology is des-cribed from serial sections of two proventriculi from birds 3 weeks 9 months of age while the histology of the infected organ is based on sections of 9 proventriculi collected at selected stages of the infection. The Normal Proventriculus Morphology (Fig. 1, 2, 4-) The proventriculus of the blue grouse l i e s to the left side of the median line at a level immediately posterior to that of the heart. It forms a bulbous expansion at the posterior end of the lower esophagus and leads, via the short, constricted interventricular region, to the gizzard. The proventriculus is firm in texture, smooth in out-line, and generally pale pink to rosy in colour. The mucosal epithelium is covered by a thin layer of mucous. Removal of this mucous by gentle scraping reveals the plicae and sulci of mucosal epithelium and the slight eminences on which are situated the mouths of the deep glands of the mucosa. Histology (Fig. 6, 7, 8, 9) There is no abrupt transition between the esophagus and the proventriculus. The stratified - 26 -§i squamous epithelium lining the esophagus covers the anterior quarter of the proventriculus. The tunica propria of this area contains the mucous glands of the esophagus while deeper in the mucosa, the anterior extremities of the deep glands of the proventriculus can be seen. The squamous epithelium gives way to the columnar epithelium which lines the remainder of the proventricular lumen. This mucosal epithelium is arranged in foliate papillae which converge to form concentric plicae and sulci around the openings of the deep glands. These gland open-ings are on slight eminences and occur mainly in the posterior half of the proventriculus becoming most numerous near the junction of the proventriculus and interventriculus. The muc-osal epithelium consists entirely of columnar mucous secreting cells with no goblet c e l l s . The distal third of these cells stained pink with the periodic acid- Schiff technique indicating the presence of a mucoid substance. The tunica propria extends into the plicae, between the deep glands and the mucosal epith-elium, and between adjacent deep glands. The muscularis mucosae is represented by longitudinal muscle fibres in the tunica prop-r i a between the deep glands and the mucosal epithelium. Muscle fibres of the muscularis mucosae also extend between and beneath the deep glands. The deep glands are large and lobulated. The secretory tubules are simple and radiate out from the central collecting ducts which are lined with columnar, mucous secreting epithelium. The apocrine cells lining the gland tubules are cuboidal, deeply eosinophilic, with a spherical nucleus. The dis t a l half of each c e l l is not attached to the neighbouring - 2? -cel l s giving the gland tubules a feathery appearance. The central collecting cavities of each gland lobule open into a wide common duct which disgorges into the lumen of the pro-ventriculus . The submucosa of the proventriculus is extremely thin and appears to be absent in places. The lamina muscularis is com-posed of an inner layer of longitudinal muscles and an outer layer of circular and oblique muscles. The serosa is thin. At the interventricular junction there is a gradual change from mucous secreting columnar cells to the columnar cells which secrete the koilin lining of the interventriculus and gizzard. The Infected Proventriculus Morphology (Pig. 1, 3, 4, 5) The changes in the external appearance of the infected proventiculus are related to the number of worms present. In general, proventriculi harbouring less than 10 worms show no external evidence of the infection. Where more than 10 worms are present, there is always an en-largement of the affected organ. Where several hundred worms are present, the proventriculus may be as large as, or consider-ably larger than, the gizzard. The largest proventriculus ex-amined contained approximately one thousand nematodes (844 counted from three-quarters of the proventriculus and the re-maining portion preserved intact for sectioning). The circum-ference of this proventriculus (5i inches) represented a three-fold increase over the^infected proventricular circumference (Pig. 1, 3). When more than 10 nematodes are present, the proventriculus may appear flabby, and a creamy white or grayish mottling is - 28 -visible on the serosal surface. On opening the organ, i t can be seen that this mottling is directly under the infected portion of the mucosa. The deep glands in this area are occlud-ed and engorged with secretory products thus becoming visible through the serosa and lamina muscularis. The typical lesion is immediately apparent as a tumour-like papillomatous out-growth on the mucosal epithelium (Pig. 4, 5). Copious glairy mucous is associated with the lesion and small hemorrhagic areas may be evident. If few worms are present, the lesion i s l o c a l -ized and discrete; however, i f several hundred worms are present, the entire mucosal surface is involved. The nematodes may not be immediately evident, but clearing away of the mucous and part-ing the papillomatous strands soon reveals the worms with their heads deeply embedded in the mass. In heavy infections, the interventriculus is enlarged and the k o i l i n lining is thickened. This lining, which normally forms a thin confluent sheet, readily fragments into asbestos-like splinters in infected material. Blue grouse dying from D. nasuta infections frequently (approximately 50% of a l l cases) exhibit intussusception in the proventricular region. Either the posterior part of the esoph-agus is telescoped into the proventriculus, or more frequently, the proventriculus is telescoped into the gizzard. It was evident, in most cases, that this condition had existed several days prior to the death of the bird and was not, therefore, a result of a convulsive death. Probably progressive weakening of the musculature, and the strong contractions required to force food past the obstructing lesion produced this condition. - 29 -Relationship of D. nasuta to the Host Tissues (Fig. 5, 10) Second, third, and fourth stage larvae, and adult D. nasuta have a l l been recovered from the mucosal epithelium of one proventriculus. As noted in the previous section there is no larval migration in the host tissues of the type illustrated by Ascaris lumbricoldes and maturation of the larvae takes place in the lumen of the proventriculus. The larva is situated in the sulci of the mucosal epithel-ium between the openings of the deep glands. In sectioned material, i t can be seen that the head just penetrates the epith-elium and lie s in the tunica propria, (Fig. 10) while the body of the larva l i e s free in the proventricular lumen. The worms attach to the mucosa in close proximity to each other forming a single lesion. Multiple lesions were never observed. Adult worms are firmly anchored by their cephalic cordons in the papillomatous tissue. If, however, several hours elapse between death and autopsy of the host, the worms may be found lying free in the proventriculus or gizzard. This seems to i n -dicate that the nematodes are not fixed in the tissues of the lesion and can move about at w i l l . Adult JJ. nasuta were found in greatest numbers around the periphery of the papillomatous growth. Where these worms i^ere established, the tissue reaction resembled that found in a rec-ently infected proventriculus. There seems to be a tendency for D. nasuta to migrate outwards from the center of the lesion where the greatest tissue reaction has occurred. Thus the worms are constantly impinging upon new areas of the proventriculus and causing the diameter of the lesion to continuously increase. - 30 -This view is further substantiated by the large size of lesions of long standing found in birds to which only a few (less than 10) larvae had been administered. Development of the Proventricular Lesion One Week Post Infection (Fig. 10, 11, 13, 14) The larvae were found with their heads embedded in the tunica propria at the bottom of sulci in the mucosal epithelium. They were con-sidered to be 4th stage larvae on the basis of the extent of development of their cephalic cordons. In the tissues surround-ing the head there were a few enlarged blood vessels, but no cellular i n f i l t r a t i o n , v e i t h e r in the adjacent plicae or in the tunica propria around the head (Fig. 10). However, there was massive cellular i n f i l t r a t i o n in the tunica propria of the bases of the plicae and around the mouths of the deep glands immediat-ely beyond the tissues in contact with the worm. The distal ends of the plicae were swollen and edematous (Fig. 13). The c e l l -ular i n f i l t r a t i o n was composed of lymphocytes (mainly small), plasma ce l l s , and an occasional granulocyte. Tissue granulocytes a l l appeared to have rounded eosino-p h i l i c granules and resembled eosinophils except for the presence of a lobed nucleus. Lucas and Jamroz (I96I) state that the spindle-shaped eosinophilic granules of heterophils become round in extravasated blood. Probably most of the granulocytes found in the tissues of the lesion were heterophils. Two Weeks Post Infection (Fig. 11, 15, 16) Sections of non-ovigerous adult worms were seen in the tissues. The tunica - 31 -propria immediately surrounding the point of attachment of the worms was very edematous but there was no round c e l l i n f i l t r a -tion in this area (Pig. 15). In adjacent areas, round c e l l i n f i l t r a t i o n was present in the plicae (particularly the prox-imal half of each), in the tunica propria, between the deep glands and between the tubular elements of these glands. Round c e l l i n f i l t r a t i o n was also present in the tunica propria between the mucous glands of the esophagus. Petechial hemorrhages have occurred in the tunica propria underlying the mucosal epithelium of the proventriculus. Three Weeks Post Infection (Pig. 17, 1 8 ) The tunica propria of the plicae was very edematous, swollen, and showed fibroblas-t i c activity. The plicae were becoming longer and broader. Fewer round cells were distinguishable in the areas of cellular i n f i l t r a t i o n and very few granulocytes were present. It was thought that much of the swelling in the plicae might have been due to an excessive accumulation of connective tissue ground substance. However, Hale and P.A.S. stains showed this not to be so. Swelling, therefore, was due to continued edema. As the age of the lesion increased, the cellular i n f i l t r a t i o n changed in character. Plasma ce l l s , monocytes and fibroblasts predominated at this time and a gradual fibrosis of the swollen plicae was observed. The cells lining the mucous glands in the posterior portion of the esophagus have become cuboidal rather than columnar. There was much round c e l l i n f i l t r a t i o n in the tunica propria be-tween these glands and in the interventriculus. - 32 -Four Weeks Post Infection (Fig. 19) At 4- weeks, the tissues were very similar to those of the 3 week stage except for the increased length and complexity of the plicae. Some giant cells were present in the tunica propria adjacent to the nematodes. Cystic mucous glands were observed in the esophagus under a thickened squamous epithelium. The tubular glands of the interventriculus had lengthened and showed much round c e l l i n f i l t r a t i o n . Eight Weeks Post Infection (Fig. 11) The lengthening and branching of the plicae had progressed and fibrosis was contin-uing. Very few round cells were present but numerous granulocytes were distinguished in the plicae and in the tunica propria of the interventriculus. Twelve Weeks Post Infection (Fig. 12, 20, 21) The long complex plicae on broad connective tissue stalks formed typical inflammatory polyps or papillomas. These papillomas showed a marked acellular fibrosis of their connective tissue cores. There were focal collections of granulocytes and a few lymphocytes. Fibroblastic activity had ceased. The covering mucosal epithel-ium appeared normal. The deep glands, particularly in the posterior part of the proventriculus, may be collapsed or atrophied with subsequent sloughing of the apocrine chief c e l l s . Connective tissue may i n f i l t r a t e between the secretory tubules of the deep glands and the mucous secreting epithelium lining the collecting ducts of the deep glands may show hyperplastic growth. The lamina - 33 -musoularis is hypertrophied. The increase can be observed in both the circular and longitudinal muscle layers although i t is more marked in the latter. In the esophageal region, cystic and engorged mucous glands are present and, in severe infections, papillomatous outgrowths of the squamous epithelium are seen. The tubular glands of the interventriculus are very long. The gland ce l l s are short, almost squamous in appearance, and their secretion appears granular. The strands of koilin found in this region tend to remain distinct and do not fuse to form a confluent sheath. Hemorrhages in the Lesion Streaks of brown or red coloured mucous were frequently found in the proventriculus of infected blue and ruffed grouse and also in the chicken. This material was submitted to the benzidine test for occult blood and proved positive. Bleeding from the proventriculus of the chicken occurred by the 4-th day of infection. In the blue and ruffed grouse, bleeding was not observed u n t i l at least 3 weeks after i n i t i a l infection. In the latter hosts, the bleeding is local and slight, occurring only at a site where several worms are established. Attempts were made to analyse the feces of infected blue grouse using the benzidine test for occult blood. A l l tests made on feces of both infected and control birds proved positive. A check soon revealed that the mash diet of the birds contained both meat scrap and blood meal and was highly positive. To - 34 -avoid, the effect of the food, the birds were starved for several hours and then fed only green vegetable matter. Feces recovered in this fashion from control and infected birds were a l l negative for occult blood. This test was made twice on 3 infected and 2 controls each time. Since weight gain and hematological data were concurrent-ly obtained from the same birds, i t was not considered advisable to deprive them of food and submit them to the additional hand-ling necessitated by continuation of these experiments. - 3r5 " Figures 1-5 Morphology of normal and. infected proventriculi from birds 12 weeks of age. Fig. 1. Esophagus, proventriculus, interventriculus and gizzard. Infected (approximately 1000 worms) #52, normal #13. X 9/20 Fig. 2. Mucosal surface of normal proventriculus. Note relative thicknesses of superficial epithelium and deep glands of the mucosa. X 1,3 Fig. 3. Infected proventriculus; 0-1 (on inch scale of ruler) is gizzard, 1-2 is interventriculus, 2-31-is proventriculus, 3i-4- is esophagus. X 4/5 Fig. 4. Infected proventriculus plus gizzard on l e f t , normal proventriculus on right. Note relative thicknesses of tissue layers in the two-proven-t r i c u l i . X 9/20 Fig. 5- Enlargement of infected proventriculus in Fig. 4. showing papillomatous tissue (white) and adult p. nasuta colled in the tissue. X 1,3 - 36 -Figures 6 - 9 Histology of the normal proventriculus. Fig. 6. Plicae and sulci of the mucosal epithelium above with deep glands of the mucosa below. X 35 Fig. 7. Mucous glands and squamous epithelium of esopha-gus overlying the deep glands in the anterior portion of the proventriculus. X 14 Fig. 8 Section through middle portion of the proventri-culus showing the arrangement and relative thick-nesses of the mucosal epithelium, deep glands of the mucosa and the muscular tunics. X 14 Fig. 9 Arrangement of apocrine cells of the deep glands and the muscular tunics. X 55 Figures 10 - 21 Histology of the infected proventriculus. Fig. 10. One week after infection. Head of 4th stage larva (arrow) has penetrated the epithelium and l i e s in the tunica propria (cross-sections of body of larva l i e in lumen of proventriculus above). Note lack of cellular exudate in vicinity of worm a l -though the tissues are edematous. Copious cellu-lar exudate is present in the plicae beyond worm and •below-and- between the deep glands. X 35 - 37 -Pig. 11 Longitudinal sections of whole proventriculi. Upper l e f t , 1 week after infection. Upper right, 2 weeks after infection. Below, 8 weeks after infection. Note increase in the length of the plicae of the mucosal epithelium. X 3 Pig. 12. Longitudinal section of whole proventriculus 12 weeks after infection. Note elongated plicae, papillomatous change of esophageal epithelium with cystic mucous glands, thickened and frag-mented koilin of the interventriculus, atrophy of the deep glands and hypertrophy of the muscular tunics. X 3 Pig. 13. One week after infection. Plicae of mucosal epi-thelium showing copious cellular exudate in prox-imal region and edema in distal region. X 140 Pig. 14. One week after infection. Cellular exudate In plicae shoxuing lymphocytes. X 360 - 38 -Pig. 15. Two weeks after infection. Worm present (arrow). Plicae have started to elongate. Cellular ex-udate in proximal portions of plicae but not in the vicinity of the worm. X 35 Pig. 16. Two weeks after infection. Section of a plica showing extravasated lymphocytes and increase in connective tissue. Fibroblasts can be seen. X 360 Pig. 17. Three weeks after infection. Further elongation and branching of the plicae. Note thickening of the tunica propria between the deep glands and the mucosal epithelium and cellular i n f i l t r a t i o n in the proximal portions of the plicae. X 35 - 39 -Pig. 18. Three weeks after infection. Fibroblasts forming in the plicae. X 280 Fig. 19. Pour weeks after infection. Further elongation and branching of the plicae. However, plicae in immediate vicinity of head of worm (arrow) have not elongated. X 35 Fig. 20. Twelve weeks after infection. Squamous papilloma of the esophagus. Note cystic mucous glands (arrow). X 35 Fig. 21. Twelve weeks after infection. Portion of the enormously elongated and complexely branched plicae forming the papilloma of the mucosal epithelium. Note the abundant fibrous connective tissue in the tunica propria of the plicae. X 4-0 - 40 -Discussion Comparison of the arrangement of tissues in the proven-triculus of the "blue grouse with that of the chicken described by Calhoun (1954) revealed no essential differences. Except for increase in size of this organ, no changes due to age were observed. Calhoun (195 )^ reported an increase in elastic t i s -sue in the proventriculus with increase in age. However, spec-i f i c stains for elastic tissue were not used in this study. The function of the deep glands of the proventriculus is similar to that of the gastric glands of the mammalian stomach. "In the mammal, pepsinogen granules are secreted by the chief cells and hydrochloric acid by the parietal cells of the gastric glands, but in birds both are secreted by the chief cells. The bird has no cells comparable to parietal cells of mammals." (Sturkie, 1954; p. 166-167). Food particles are seldom found in the lumen of the pro-ventriculus indicating that the passage of food through this organ is rapid. This fact coupled with the information that the pH of this organ is higher than the optimum pH for peptic digestion suggests that l i t t l e i f any digestion i s accomplished in this organ (Sturkie, 1954). Whether or not the proventri-culus and i t s secretions are essential for the survival of the bird are not known. However, i t is reasonable to presume that an infection causing severe damage to this organ would serious-ly affect the digestive capabilities of the bird. Hwang _et a l , ( I 9 6 I ) reported that the worms were embedded in mucosa but had not penetrated to the "muscular tunics". This statement is not - 41 -entirely clear. However, from their illustrations, and because they use the tissue designations suggested by Calhoun (1954), i t appears that the statement means that the head of the worm is embedded in the mucosal epithelium but has not penetrated as far as the muscularis mucosa. Wasielewski and Wulker (1919) also, are not clear on this point. They state that the worms are embedded in the tumourous mass. They do not indicate invasion of the deeper layers of the mucosa by the worms. On the other hand, Edminster, (1947) states that, m severe cases, among ruffed grouse, the worms penetrate and des-troy the gastric glands (deep glands of the mucosa?) and that perforations of the proventriculus can occur. In the present study, several hundred blue grouse proventriculi infected with 1 to approximately 1000 D. nasuta were examined macroscopically. In a l l of them, the worms had never penetrated deeper than the superficial mucosal epithelium. The histological examination of infected blue grouse pro-ventriculi revealed no evidence of necrosis as described by Cram (1928) for the pigeon, or ulceration as mentioned by Wehr (1948) for ruffed grouse. Nor do the tissue layers become in -distinguishable as described by the latter author. Evidence of secondary bacterial invasion (Hwang et a l , I96I) was also lack-ing. Desquamation as described by these authors was found only in the deep glands of blue grouse with very severe infections. Sloughing of the apocrine cells in these glands cannot be at-tributed to invasion of these glands by the worms. Probably desquammation in these glands is a result of occlusion of the of the gland opening and pressure effects. - 42 -Hwang et a l (1961) found minimal inflammatory c e l l reaction. 11 Wasielewski and Wulker (1919) reported the absence of hemor-hagic alterations, leucocyte accumulations and inflammatory gran-ulation tissue. The present study has shown that cellular in-f i l t r a t i o n reaches a peak during the f i r s t 3 weeks of infection. As the age of the lesion,progresses, the cellular i n f i l t r a t i o n changes in character, fibroblast activity with progressive fibrosis is evident, and there is a gradual accumulation of granulocytes in the tissues. At 12 weeks, the fibroblastic activity in the papillama appears to have ceased despite the presence of the worm. Perhaps* by this time, the t i t r e of antibody in the tissues surrounding the worm-is sufficiently high to neutralize the antigens of the worm thus allowing the tissues to become quiescent. Alternat-ively, the extravasated lymphocytes which accumulate in the tissues unti l 3 weeks after infection may give rise to the f i b -roblasts which ultimately form the connective tissue cores of the papillomas. By 12 weeks, when fibrosis ceases, most of these lymphocytes have disappeared from the tissues. (The mutability of lymphocytes was described by Roberts in i960.) The peculiar lack of tissue reaction in the immediate vicinity of the worms is interesting. No satisfactory explan-ation can be offered for this phenomenon. The presence of an acellular material around the heads of invading worms has been reported for Stammerlnema soricis (formerly Dispharvnx soricis) in a shrew by Tiner (1951) and by 11 Wasielewski and Wulker (1919) for D. nasuta in the pigeon. - 43 -Slides of infected, shrew stomachs, prepared "by Tiner , were examined to ascertain the nature of this exudate. A reaction of this type does not occur in the blue grouse where the only tissue change in the vicinity of the head of the worm is the formation of giant c e l l s . The only allusion to the presence of hemorrhages in the lesion is that made by Edminster (1947) who referred to " a small bloody lesion of the epithelial tissue". Bleeding from the proventriculus of infected blue and ruffed grouse is evident from examination of the lesion. However, the limited series of tests for occult blood in the faeces were insufficient to i l l u s -trate this phenomenon. The benzidine test is highly sensitive and probably continued testing would have revealed the presence of blood in the faeces of infected birds. Examination of proventricular lesions of naturally infected birds at game checking stations showed that more hemorrhagic foci were present in the ruffed grouse than the blue grouse when comparable numbers of p. nasuta were present. Although the hemorrhagic foci are never extensive, they represent areas where there is continuous blood loss. The significance of these hemorrhages on the hematology of the bird w i l l be discussed later. * Tiner's slides were kindly loaned to me by Dr. R. Rausch of the United States Department of Public Health, Anchorage, Alaska. _ 2J4 -Section III EFFECT OF THE PARASITE ON THE HEMATOLOGY OF THE HOST Introduction Although Cram (1928) suspected anemia from the pale colour of the i r i s of infected pigeons, no hematological studies have been performed on birds infected with D. nasuta. It has been established by Fantham (1910), Allen and Gross (1926), and Olsen and Levine (1939) and many others, that nema-tode infections can produce fluctuations in the hemoglobin level and changes in the differential blood c e l l counts of affected birds. To test the validity of Cram's (1928) statement and to correlate the development of the proventricular lesion and of the worm with changes in differential blood c e l l counts, two procedures were used. In addition, the hematology of a series of control birds was established as a standard for comparison. Materials and Methods The hematological standards for control birds are based on samples taken from 10 birds ranging in age from 14 to 99 days. The two procedures used with infected birds are: (a) D. nasuta larvae were administered to chicks in a single large infective dose. This method was used in order to cla r i f y the results so that changes in the hematology would not be affected by subsequent larval administrations. Two chicks were given 100 larvae each at 30 days of age. - 45 -At autopsy these chicks had 64 and 42 adult p. nasuta in the proventriculus. One chick was given 200 larvae at 30 days of age and harboured 126 p. nasuta at autopsy. Blood was sampled on day 0 immediately before adminis-tration of larvae and at 3 day intervals thereafter for 30 days. (b) Chicks were given 10 to 20 larvae on consecutive or alternate days over a period of one week until 20 to 100 larvae were presented. This procedure approximates the method by which infections are acquired under natural conditions. Blood samples were taken from 12 infected birds which on autopsy were found to have from 6 to 22 worms. Sampling was conducted from day 0 to day 86 at roughly weekly intervals. In some cases, samples were taken twice weekly during the i n i t i a l stages of infection. For each of these procedures, data for day 0 were plotted separately. Subsequent samples were plotted according to the elapsed time interval after i n i t i a l administration of larvae. Samples f a l l i n g within 4 day intervals (i.e. 0-3, 4-7...84-87 days) were grouped and plotted at the mid-point of the respect-ive interval. The Hematology of Control Birds (Fig. 22) Plots (not presented) of the number of erythrocytes, poly-chromatic erythrocytes and leucocytes per cu. mm. of blood and of the grams of hemoglobin per 100 cc. of blood showed no d i f -ferences due to age or sex of the chicks. Birds 14 to 35 days - 46 -of age had erythrocyte counts and hemoglobin values comparable to those of birds 80 to 99 days old. Means and ranges of values for erythrocytes, polychromatic erythrocytes, leucocj/tes and hemoglobin obtained from control blue grouse are given in Table III. The means and extremes of range for the various leucocytes in these birds are presented in Table IV. When stained with Wright's blood stain, the cellular e l -ements of the blood of the blue grouse may be identified on the basis of the following descriptions. Cell measurements are not given because the variation in c e l l sizes is only in part actual. Much of i t is a function of smearing technique. Erythrocytes These cells are oval, with eosinophilic cy-toplasm. The centrally located nucleus is oval and dense in mature forms but is round in younger forms. The chromatin is blocky and stains dark blue-purple. Polychromatic erythrocytes These cel l s , which are broadly oval in outline, have basophilic cytoplasm and round nuclei with reticular chromatin. Early polychromatic erythrocytes were frequently encountered. They were smaller, round in outline and more basophilic, with larger, more openly reticular nuclei. When the count of mature erythrocytes was low and the count of polychromatic erythrocytes wery high (in infected birds only) these cells were found in mitosis. Dividing cells were usually in metaphase, or rarely, telophase. The chromosomes of these cells stained dark blue and the cytoplasm was a uniform grey-blue. Thrombocytes In smears of blood not treated with an - 47 -anticoagulant, disintegrated, thrombocytes appear as pink staining patches. They can be readily distinguished from the occasional ruptured erythrocyte by the reticular border of the patch and the lack of nucleus. The dispersed cytoplasm of ruptured erythrocytes tends to round up, and the nucleus, which usually swells, maintains i t s identity. Heterophils These cells are relatively numerous in the blood. The nucleus is polymorphic, usually displaying 2 and occasionally 3 lobes. These lobes stain pale blue and exhibit blocky chromatin. The cytoplasm is clear and contains many rod-shaped eosinophilic granules. Eosinophils The c e l l s , so categorized, have densely staining, dark blue, round or oval nuclei. The cytoplasm is slightly basophilic. These cells are densely packed with round, refractile, eosinophilic granules. The nucleus is always loc-ated at the periphery of the c e l l , and forms a small bulge in i t s outline. These cells are scarce - never forming more than \% of the leucocytes of control birds, nor more than 3% in in-fected birds. Occasionally, cells of this type possess a kidney-shaped nucleus and oval granules. Basophils The nucleus of these cells stains pale blue and may be oval or kidney-shaped. The granules are large, round, and densely basophilic. These cells are rarely seen. Monocytes The nucleus of the monocyte is oval or kidney-shaped and is situated at the periphery of the c e l l . The nucleus stains red-violet while the cytoplasm is frequently grey or very weakly basophilic. Clear vacuoles occur in the - 48 -cytoplasm. On occasion these vacuoles take on a dense "basophilic stain giving the c e l l the appearance of a baso-p h i l . The red-violet colour of the nucleus, however, identi-fies such ce l l s as monocytes. Lymphocytes Preliminary examination of blood films from infected grouse indicated changes in the abundance of lympho-cytes of different sizes. Lymphocytes were separable into two groups designated as large lymphocytes and small lymphocytes. The small lymphocyte has a small amount of clear to slightly basophilic cytoplasm. The nucleus is round, compact and stains deep blue. Minute eosinophilic granules were occasionally ob-served adjacent to the nucleus. The nucleus of the large lymphocyte is blue-violet and has a blocky arrangement of the chromatin. The cytoplasm which may be scant or plent i f u l , is basophilic, and concentrations of minute basophilic granules may be seen at the c e l l boundaries. These cells are frequently very irregular in outline, conforming to the contours of adjacent c e l l s . In thin smears, or when isolated from neighbouring cel l s , large lymphocytes are round, with numerous pseudopod-like extensions of the cytoplasm. The Hematology of Infected Birds Graphical representations of the various blood components studied in infected birds are given in Figures 23, 24, and 25. Agglutinated erythrocytes, in groups of 3 to 8, were ob-served in the circulating blood of two birds sampled on day after administration of larvae. This phenomenon was never ob-served again although samples were later taken at the same interval from other birds. - 49 -Figure 22 Hemocytes of the blue grouse Key to Illustrated Blood Cells 1. Eosinophil with small granules. 2 . Eosinophil with large and small granules. 3. Heterophil. 4. Small lymphocyte. 5 . Large lymphocyte. 6. Polychromatic erythrocyte in mitosis (metaphase). 7. Monocyte. 8. Polychromatic erythrocyte. 9. Erythrocyte. - 50 -Table III Means and ranges of values for erythrocytes, polychromatic erythrocytes, leucocytes and hemoglobin obtained for control blue grouse. Blood characteristic Sample size Mean Range Erythrocytes, millions per cu. mm. 25 2.61 2.00-3.04 Polychromatic erythrocytes, thousands per cu. mm. 25 60.9 25.0-91.0 Leucocytes, thousands per cu. mm. 25 45.7 23.8-77.8 Hemoglobin, grams per 100 cc. 20 11.2 10.0-13.1 Table IV Means and ranges, for control blue various leucocytes per cu. mm. of grouse, of values for blood. Leucocyte Sample size Mean Range Heterophils 23 7,500 3,400-15,900 Eosinophils 23 0-779 Basophils 23 0-392 Monocytes 23 1,026 572-1,9^6 Total lymphocytes 23 3 ,^950 19,754-48,115 Small lymphocytes 23 28,000 13,923-35,264 Large lymphocytes 23 9,028 5,589-12,851 - 51 -Figures 23, 2k, 2$. Variation in the numbers of certain hem-ocytes per cu. mm. of blood related to the time elapsed after infection. Open symbols represent birds given large single doses of worms (procedure a). Solid symbols repres-ent birds given repeated small doses of worms (procedure b). The continuous horiz-ontal line represents the mean value for uninfected control birds. The dotted horizontal lines represent the upper and lower limits of the range of values in un-infected birds. Note the differences in the vertical scales. Fig. 23. Relationship between the numbers of certain hemocytes present in the blood of infected blue grouse and the time elapsed after in-fection. A. erythrocytes, B. polychromatic erythrocytes, and C. leucocytes. 3 5 - A ERYTHROCYTES co 2 2 5 V 3.Q4J 2.61 8 o m 3 O 1 . 5 -V 2 00 O PROCEDURE A • PROCEDURE B B POLYCHROMATIC ERYTHROCYTES K 0 2 Q. CO UJ »->-o o 0 I - # 91,000 6C\9bc o-q 25j000 £ 0 0 7 0 0 6 0.04 77300 C TOTAL LEUCOCYTES • 45,000 0.02 -10 20 30 40 50 60 70 DAYS AFTER INFECTION 90 100 - 52 -Fig. 24. Relationship between the numbers of certain leucocytes present in the blood, of infected blue grouse and the time elapsed after infection. A . total lymphocytes, B. small lymphocytes, and C. large lymphocytes. o o o _J CD U. O 3 O CC Ul CL CO UJ f->• o o Ul X or Ul CO 60,000 40,00 0 20,000 10,000 35,000 30,000 20,000 10,000 20,000 10,000 A TOTAL LYMPHOCYTES 48,145 J I I L J I L • o B SMALL LYMPHOCYTES 35,264 C LARGE LYMPHOCYTES O PROCEDURE A • PROCEDURE B J L J i _ 10 20 30 40 50 60 70 80 90 DAYS AFTER INITIAL INFECTION - 53 -Fig. 25. Relationship between the numbers of certain leucocytes present in the blood of infected, blue grouse and the time elapsed after infection. A. heterophils, B. eo-sinophils and C. monocytes. o o 3 m u. o 20,000 15,000 10,000 5,000 3 U £ 1,500 CO Ul £ 1,000 u o ui 500 x tr ui co 1 2,000 z 1,000 A HETEROPHILS B EOSINOPHILS 779 211 J I I L C MONOCYTES 1,946 J I I L J L O PROCEDURE A • PROCEDURE B J l L 10 20 30 40 50 60 70 80 90 100 DAYS AFTER INFECTION - 5 4 -Discussion A notable feature of the blood was i t s variability. Relatively large fluctuations in erythrocyte, polychromatic erythrocytes, leucocyte and hemoglobin values were obtained between individuals and between weekly samples from the same individual. There is a considerable variation in the blood picture of the normal fowl (Palmer and Biely, 1935a) and (Sturkie, 1954) suggests that caution should be exercised before att r i b -uting changes in the blood picture to disease. Cook (1937) comments on the sensitivity of the white blood c e l l system of chickens to dietary deficiencies or toxic substances. Variability seems to be an important characteristic of avian blood histology. Some investigators have attempted to discount this feature on the basis of faulty techniques. To a certain extent, this is probably valid, and certainly confusion in identification of the various c e l l types, particularly throm-bocytes (Blaine, 1928) has arisen. Throughout the study, control blue grouse chicks exhibited a high leucocyte count. Comparison with reported leucocyte levels for chickens show that the levels obtained for the blue grouse may be abnormally elevated. Olson and Levine (1939) reported that an unknown factor or factors caused leucocytosis in the entire group of chickens sampled. Cook and Scott (1935a, 1935b) and Cook (1937) proved experimentally that some types of f i s h meals used as the protein source in poultry diets produced a marked increase in the leucocyte count, which per-sisted as long as the diet was administered. Pish meal accounted - 55 -for a portion of the available protein in the commercial diet fed to the grouse chicks and may be responsible for the apparent leucocytosis. The confinement of the chicks may also account for this feature. Palmer and Biely (1935"b) found, that the blood of confined chickens showed elevated leucocyte levels and depressed erythrocyte counts. Olson and Levine (1939) found that the factors of age and sex did not influence the count of leucocytes of normal chickens up to the age of 196 days. Twisselman (1939) found no di f f e r -ence in the leucocyte and erythrocyte counts in white leghorns from 4-7 days of age to maturity. However, Cook (1937) states that there is a tendency for increase of hemoglobin values and erythrocyte counts in white leghorn chicks up to 85 days of age. The blue grouse chicks did not show any changes in the blood elements which could be attributed to age or sex. The eosinophils of the blue and ruffed grouse resemble the eosinophilic metamyelocyte of the chicken or the eosinophils of the turkey as illustrated by Lucas and Jamroz (I96I). Eosino-phils with lobed nuclei as described from the chicken by Olson (1937, 1948), Porkner (1929), Lucas and Jamroz (1961) and from the "grouse" (red grouse?) by Fantham (1910) were never encoun-tered although approximately 75,000 leucocytes were examined from control and infected blue and ruffed grouse. The values of the various hemocytes obtained for uninfected blue grouse are presented as control values and do not neces-sarily represent the normal hematology of the blue grouse. Because of the sensitivity of the avian blood system, these - 56 -values are only valid, for comparison with those of birds raised under comparable conditions of diet and confinement. The erythrocyte counts of infected birds are generally lower than those of the control birds, -^ t is evident (Pig. 23) that after administration of larvae, there is a sharp decline in the number of erythrocytes. A minimum level, reached by the third day after infection, may represent a loss of approximately one-third of the red blood cells (erythrocytes and polychromatic erythrocytes) present immediately before infection. The loss of mature erythrocytes is actually greater than the above figure since at their lowest point, approximately 10-20$ of the ery-throcytes present are polychromatic. The hemoglobin levels, as would be expected, follow the same pattern as the erythrocyte values. However, because of the large numbers of polychromatic erythrocytes in the circulating blood of infected grouse, the minimum hemoglobin levels are much farther below those of the control birds than are erythrocyte minima. The polychromatic erythrocytes (Pig. 23) form a curve which is roughly a mirror image of thati-obtained for the erythrocytes. There i s , however, a considerable lag in phase and a tendency toward overcompensation. This i s probably a function which depends on the hemoglobin levels in the blood. Hypoxia, a result of low hemoglobin level, stimulates the hemopoetic centers to produce and release into the circulating blood large numbers of immature erythrocytes. By the time these cells have matured, and the hemoglobin level has risen sufficiently to depress erythrocyte - 57 -formation, there has been an overproduction of these cells. At the peaks of polychromatic erythrocyte levels, divid-ing forms were found in the circulating blood. They were found in numbers representing 0.02$ of the total erythrocytes present, or 400 to 600 mitotic polychromatic erythrocytes per cu. mm. of blood. The presence of these cells in circulating blood is interpreted as a shift to the l e f t on the Schilling scale and indicative of extensive erythrocyte loss and active regeneration of these c e l l s . Subsequent to infection (both procedures), the immediate loss of erythrocytes, compensatory rise in polychromatic erythrocytes and correlated drop in hemoglobin values suggest that the invading larvae produce a substance which damages the red blood c e l l s . This view is suggested by the presence of agglutinated erythrocytes in the circulating blood, and by the absence of hemorrhages during the f i r s t two weeks of lesion development. There i s , as well, no evidence that D. nasuta ingests blood. The second decrease in erythrocyte numbers occurring 18 to 22 days after i n i t i a l infection is more d i f f i c u l t to explain. It is possible that i t .can be correlated to harmful substances released during the moult of the 4th stage larvae which is completed by day 13. The decrease is too sudden to be explained by the effect of the developing papilloma on the digestive and assimilative capacities of the bird. It may, however, result from the onset of local hemorrhages in the proventriculus. The hemorrhages may, in turn, result from increased activity of the - 58 -worms as they approach maturity - small migrations as males seek out females - with resultant focal concentrations of several worms. Leucocyte levels (Fig. 24) are mainly affected by the numbers of lymphocytes which form 70 to 90$ of the total leucocyte count. Small lymphocytes form 45 to 70$ of the total leucocyte count. From figure 24, i t can be seen that during the course of the infection, the relationship in number between large and small lymphocytes may change considerably. The ratio of small to large lymphocytes in control birds ranged between 4.4:1 and 2.4:1. In infected birds this ratio ranged between 9.1:1 and 1.7:1. The lower ratio may be due to the rapidity with which the small lymphocytes leave the circulating blood to accumulate at the site of infection (indicated by studies on the development of the lesion). The higher ratio may result from the rapid production and maturation of small lymphocytes in response to their removal from the circulating blood. Leucocytes tend to show the same pattern of fluctuations as do the erythrocytes. A leucocytopenia evident immediately after infection is followed by a leucocytosis which reaches a peak between the 5th and 10th days of the infection. Subseq-uently, leucocyte counts return to normal or subnormal levels only to rise to a second peak approximately 40 days after i n -fection. This general pattern is followed by the lymphocytes. Heterophils, eosinophils and monocytes (Fig. 25) also show an i n i t i a l decrease and a peak between 5 and 10 days after infect-ion. However, there is considerable variation in their - 59 -subsequent fluctuations. The early leucocytopenia is probably a response to the inflammatory condition in the proventriculus with resultant extravasation of these ce l l s . In this respect, i t may be noted that the decrease in heterophils is slight. Studies on the development of the lesion show that the numbers of granulocytes found in these tissues increase very gradually and that they are infrequently encountered in the f i r s t two weeks of the in-fection. They are, however, very numerous in the tissues of the mature lesion. The subsequent rise in leucocytes is probably, in part, a direct response to the presence of the infecting agent and, in part, a response to the loss of these cells from the c i r c u l -ating blood. The second decline in numbers of circulating leucocytes is of greater magnitude than the f i r s t (procedure a). The reasons for this decrease, which begins 10 days after in-fection, are obscure. Several explanations may be offered. It may result from depression of the hemopoetic centers in response to the high levels of circulating leucocytes present at 10 days. However, this would not explain the magnitude or precipitous nature of this second decline. A tenable explanation is found when the development of the worm and i t s relationship to the tissues are considered. The f i n a l moult of D. nasuta (from 4th stage to adult) occurs between 10 and 13 days after invasion of the proventriculus. The decrease may be a response to some harmful substance in the exsheathing f l u i d of the larva or in-creased activity of the worms at this time may i r r i t a t e and inflame the tissues. This is corroborated by the observation - 60 -that the quantity of cellular exudate in the tissues of the lesion reaches a peak between the 2nd and 3rd weeks of i n -fection. It was possible to compare the hematology of birds infec-ted by procedures a and b (Pig. 23, 24, 25). There seemed to be no absolute relationship between numbers of larvae adminis-tered and magnitude of changes in relative numbers of hematocytes. Small infections may e l i c i t greater fluctuations in blood elements of some individuals than larger infections in others. It may be noted, however, that for those birds with heavier infections (procedure a), the amplitude of the fluctuations in numbers of the hematocytes is generally more extreme. For birds infected by procedure b, changes in numbers of hematocytes did not necessarily occur at the same interval after i n i t i a l infection in a l l individuals. Also, reactions were not generally as precipitous as those observed for birds infected by procedure a. This is probably directly related to the number of successful larval establishments from each infective dose. Birds infected with procedure b show a greater depression in numbers of erythrocytes and a slower recovery rate. This seems to indicate that repeated administration of a few larvae is more detrimental than the single administration of many larvae. The presence of a larval secretion capable of causing erythrocyte agglutination has been postulated. Repeated introduction of this substance by means of repeated larval administrations would then retard the formation of erythrocytes. After the i n i t i a l stages of the infection, there is a tendency for the erythrocyte numbers to remain at a subnormal - 61 -level. Leucocytosis and lymphocytosis are generally apparent. However, for the latter, the small lymphocytes remain above the mean control level while the large lymphocytes tend to remain below this level. The tendencies towards chronic heterophilia and eosinophilopenia are more marked. None of these chronic levels, which may be considered as diagnostic hematological characteristics of D. nasuta infections, are very marked. In no case do they exceed the range for con-t r o l birds. Birds suffering from larger infections would per-haps exhibit a wider deviation from the control levels. It is impossible, as yet, to use hematological evidence as diagnostic for certain types 'of helminth infections because of the paucity of comparitive data. However, in the blue grouse with chronic infections of D. nasuta there is a tendency to-wards heterophilia, eosinophilopenia, leucocytosis, lymphocytosis (small forms), lymphopenia (large forms) and anemia. 4 - 62 -Section IV EFFECTS OF THE PARASITE ON THE GROWTH AND DEVELOPMENT OF THE HOST Introduction Three aspects of the growth and development of infected blue grouse were considered; weight gain, bone chemistry and feather development. Wing, Beer and Tidyman (1944), Bendell (1955) and Stiven (1959) have studied the growth (as indicated by weight increase) of blue grouse chicks. The conditions under which their results were obtained were not s t r i c t l y comparable with those of this study. It was necessary, therefore, to establish the growth rate of control birds raised under the same conditions as those infected with D. nasuta. To assess the significance of any differences found between the weight gains of infected and con-t r o l birds, a study of the food intake of the two groups of birds was required. Signs of lameness appeared in two grouse chicks 3 to 4 weeks after administration of larvae at 1 to 2 weeks of age. Perosis was f i r s t suspected but careful examination of the tibial-metatarsal joint revealed no abnormalities in this region. Prof. J. Biely, Head of the Department of Poultry Science, Faculty of Agriculture, University of Br i t i s h Columbia, examined these birds and confirmed this negative diagnosis. The lameness progressed until the birds were unable to support their own weight and were forced to progress on the tarsometatarsus. Autopsy showed that the femurs of each bird - 63 -were bowed. In one case, the bones were soft and could be laterally compressed between the fingers. The second bird exhibited less of the bowing and the femur could not be com-pressed by d i g i t a l pressure. In one set of 5 experimental birds, 2 of the 3 infected birds suffered breakage of a wing. The control birds, which were handled as frequently, did not suffer any broken limbs. There was no excessively rough hand-ling which might have accounted for the breaks. Although some control birds suffered a broken wing or leg, these breakages could usually be accounted for by resistance on the part of the bird to the restraint necessary during the weighing or blood sampling, or by incautious fl i g h t on the part of temporarily escaped birds. It seemed possible that the bones of infected birds might be lacking in calcium, phosphorus or both. Analyses were car-ried out on femurs of experimentally infected and control blue and ruffed grouse to determine i f this were the case. It was evident from examination of naturally infected birds shot in September and of laboratory infected birds, that feathering was affected by heavy infections of D. nasuta. Ac-cordingly, a study of the effect of infection on the production of feathers was made. Materials and Methods The weights of approximately 60 grouse chicks of the two subspecies D. o. fuliginosus and D. o. pallidus were recorded. D. o. fuliginosus chicks were weighed un t i l they reached 20 - 64 -weeks of age whereas JD. o. pallidus chicks were weighed up to 12 weeks of age. The growth of male and female chicks of the two subspecies was compared to see i f they would be sufficiently similar to justify grouping of the data. Two transformations were used on the control weight data, a Walford transformation (Walford, 1946) of the weekly weight averages and a log-log transformation of a l l the weight data. The results of the latter data treatment are presented in Appendix B. These data were used as a basis for comparison with the weights of experimental blue grouse chicks, infected from 8 to 90 days of age with 10 to 200 larvae and with resulting infections of from 1 to 126 worms. The growth of a single male ruffed grouse chick infected with 11 worms at 10 days of age was recorded and compared with the growth of 18 control ruffed grouse. The femurs of 5 infected and 2 control blue grouse were analysed for calcium and phosphorus. The femurs of an infected and a control ruffed grouse were also tested. A l l the birds from which the femurs were extracted had been infected between 10 days to 4 weeks of age and had carried the infection for at least 60 days. Infection levels ranged from 3 to 22 worms. - 65 -Pig. 26. Walford transformation of weight data from 2 sexes and 2 subspecies of blue grouse chicks. The time interval is one week. I400r I i 1 1 1 1 1 i i 1 • • • 0 200 400 600 800 1000 1200 WEIGHT IN GRAMS AT TIME T - 66 -Growth Rate of Control Blue Grouse Plots of the average weekly weight values follow a sig-moid curve (Fig. 27). Using the Walford transformation of these weekly weight values, the curves approximated straight lines which could be expressed by the equation y = mx + b (Fig. 26). Comparison of the coefficients of regression, (Table V.) shows that there is no significant difference between the growth rates of the males of the two subspecies raised under the same environmental conditions (d.f. =18; t . 0.482; 0.70>p>0.60). Likewise, no significant differences were found between females of the two subspecies (d.f. = 24; t =0.232; 0.90>p>0.80), and between males and females of the two subspecies (d.f. = 21; t = 0.557; 0.70>p>0.50). Because of the lack of significant differences between the growth rates of males and females and between the two subspecies, the weights of control birds were pooled for comparison with the weights of infected birds. Table V. Values for blue grouse chicks of "m" and "b" in the linear growth equation y = mx + b using the Walford transformation of growth data. Sex Number Time (days) "m" Subspecies fuliginosus m fuliginosus f pallldus m pallidus f 15 13 13 18 3- 142 7- 143 1- 71 1- 99 0.9563 0.9464 1.0613 0.9927 89.6225 78.6215 57.9526 58.0249 - 67 -Pig. 27. Relationship between weight gain of infected blue grouse chicks and (a) the age at which the infec-tion was acquired (b) the number of worms present. The dotted lines represent the growth of infected individuals. The solid circles are the mean weekly weights of the control birds. The vertical lines are the extremes of range of weight values for control birds. The figures on the curves repres-ent the number of worms present. < or o 700 -500 300 100 0 700 5001 I 300 ui 100 0 700 500 300 100 0 A INFECTED AT 8 DAYS 1 16 WORMS ' » • I L B INFECTED AT 14 DAYS 16 21 26 C INFECTED AT 30 DAYS 42 64 126 • • 22 J L 4 5 6 7 8 9 AGE IN WEEKS 10 II 12 - 68 -Growth of Infected. Grouse The growth of the infected blue grouse is presented in Figure 27. Excluded from this figure are (a) the weights of birds infected with less than 7 worms, (b) a series of 4 birds infected at 78 and 90 days and harbouring 11 to 29 worms. In neither case (a) nor (b) did the weight values deviate from the control values. The growth of the infected ruffed grouse is presented in Figure 28. Food Intake of Control and Infected Blue Grouse The results of this study are presented in Table VI. The ratio of total food consumed to weight gained during the exper-imental period proved to be a useful indicator of the efficiency of the birds. Bone Development of Infected Blue Grouse The results of the chemical analysis performed on the femurs of grouse chicks are presented in Table VII. Examination of these results shows that the ash to dry weight ratio, per-centage phosphorus and calcium and calcium to phosphorus ratio vary as greatly within the control birds as between the control and infected birds. Wo correlation, therefore, could be found between the presence of the infection and the quantity of cal -cium and phosphorus in the bones. Feather Development of Infected Blue Grouse No differences in feather production could be observed be-tween controls and birds infected at 4 weeks of age or later. - 69 -Nor were the feathers of birds infected with less than 10 worms at or before Ik days of age noticeably affected. In birds with heavy natural infections, and those infec-ted with more than 10 worms before 2 weeks of age, the follow-ing was noted. There appears to be no affect on the growth of the primaries, secondaries, retrices and main body coverts. As well, there is no evidence of feather checking. There i s , however, retardation of the feathers in the following regions; a x i l l a r , malar, interramal, l o r a l , ocular, cervical region of the ventral and spinal tracts, anal c i r c l e t , tarsal and ventral anterior patagium. These feather, in the last tracts of the juvenal plumage to appear are generally complete by 5 weeks of age. In infected birds, these tracts may s t i l l be incomplete at 8 to 12 weeks of age. This is particularly true of the feathers of the ventral anterior patagium. - 70 -Pig. 28. Relationship between the growth of 1 9 control ruffed grouse and a single ruffed grouse infected with 1 1 D. nasuta. The vertical lines represent the limits of the range of weight values obtained for control ruffed grouse. 700 h AGE WHEN INFECTED t t t \ t t t o o • CONTROL CHICKS O INFECTED CHICK I 2 3 5 6 7 8 9 10 II 12 13 14 AGE IN WEEKS - 7 1 -Table VI Relationship between quantity of food consumed and gain in weight of infected and control blue grouse. (1) (2) (3) (4) (5) (6) (7) (8) (9) Age Age Ave. Total Wt. gain when period intake intake in (5) (7)/ inf. (days) (gm./day) (gm.) (gm.) '(8) Bird Sex (days) 1 f 0 3^-59 33.36 834.0 317.8 2.62 2 m 0 - 37-55 40.85 776.2 232.9 3.33 3 m 0 — 42-65 4Q.27 1182.4 304.0 3.89 4 f 0 - 40-55 33.62 504.3 146.3 3.45 5 f 22 8 3^-59 3 2 . 3 1 807.8 53.7 15.04 6 f 0 _ 63-78 44.42 666.3 195.6 3.41 7 f 12 3 63-78 54.81 822.2 68.2 12.06 8 f 0 _ 81-110 5 1 . 2 6 1486.5 118.6 12.53 9 f 6 77 81-110 51.83 1503.0 131.3 11.45 10 f 11 77 81-110 42.43 1230.6 101.6 12.11 11 m 0 — 81-110 63.49 1841.3 216.9 8.49 12 m 5 77 81-110 58.77 1704.4 269.O 6.34 Explanation of abbreviations used in Table VI. D. n. number of p. nasuta recovered on autopsy. Age when inf. age of chick when larvae f i r s t administered. Age period age of chick over period in which food intake and weight gain were recorded. (7)/ '(8) total food intake (in experimental period). divided by weight gain (in experimental period), grams of food required to produce a gram increase in body weight. Table VII Relationship between calcium and phosphorus in femurs of infected and control grouse chicks Bird Sex No. of Nemas Dry wt. Ash wt. Ash wt. Dry wt. % $P of Ash wt. $Ca. of Ash wt. Ca/P ratio Blue grouse, infected 1 f 7 1.5647 0.9198 58.78 17.1 25.3 1.48 2 f 7 1.2719 0.7547 59.34 16.2 28.4 1.68 3 m 7 1.4584 0.8489 58.21 13.6 26.4 1.94 4 m 3 0.9718 0.5420 55-77 12.5 21.6 1.73 5 m 22 0.6551 0.3578 54.62 13.6 29.3 2.15 Blue grouse controls 1 m 0 1.7704 1.0411 58.81 14.1 25.0 1-77 2 f 0 ,0.9271 0.4600 49.62 16.4 27.8 I.69 Ruffed grouse infected 1 m 11 0.7305 0.4289 58.71 15.7 27.8 1.77 Ruffed grouse control 1 f 0 0.8965 0.5214 58.16 15.0 36.4 2.42 - 73 -Discussion The weight gains of control blue grouse of the two sexes and. two subspecies were remarkably consistent. The means and extremes of range of the pooled weekly weights of a l l the control birds served as a basis for comparison with the weights of infected birds. Whether or not the growth of infected birds is affected depends on the age at which the infection is acquired and the number of worms present. Figure 27 shows that the growth of chicks harbouring 7 worms is not affected even though the infection was obtained at an early age (8days). Infections of 16 and 22 worms acquired at the same age seriously re-tarded the growth of the affected chicks which ultimately succumbed to the infection. Infections acquired at 14- days of age, 11 worms are just sufficient to produce a growth rate comparable to that of the slowest growing controls, while larger infections cause a deviation from the control levels. If the infection is acquired at 30 days of age, a larger number of worms (64, 126) is required to cause the growth of the infected birds to deviate from that of the controls. An infection of 42 worms at this time was not sufficient to af-fect growth although such an infection acquired at one week would probably prove fa t a l . There is a general tendency for the growth to decrease in the f i r s t week after infection. Subsequently, the growth rate increases until 3 to 5 weeks after infection when the rate again slows appreciably. The f i r s t decrease seems to be - 7^ -associated with the number of worms present, i.e. the magnitude of the depression and i t s duration are directly proportional to the numbers of worms. The second decrease occurs at approxi-mately the same time as the second c r i t i c a l hematological phase and is probably a reflection of the same causes, with the added effect of the papillomatous lesion on the digestive and assim-il a t i v e capacities of the bird. Examination of Figure 28 shows that 11 worms are suf-icient to depress the growth of the infected ruffed grouse be-low the range of the control birds 29 days after infection. This bird did not, however, show any distinct periods of growth retardation. The retarded growth rate of infected birds cannot be at-tributed to failure on the part of these birds to eat. Food intake studies (Table VI) show that infected birds consume as much as the controls. The efficiency of food conversion of chicks infected during the f i r s t two weeks of l i f e is compar-atively low - 4 to 5 times as much food is required to produce a unit gain in weight in these birds in comparison with the controls (column 9, Table VI). Birds infected at 11-g- weeks, which is near or at the end of the period of rapid growth show l i t t l e difference in efficiency from the controls. No evidence was found that the calcium:phosphorus ratio in the femurs of infected birds was disturbed although these bones were frequently bowed or broken. Studies of the mineral metabolism of nematode infected lambs (Shearer and Stewart, 1933; Franklin et a l , 1946) and rats (Rogers, 1941, 1942) showed that calcium and phosphorus were poorly u t i l i z e d . - 75 -Andrews (1938) and Andrews et a l (1944) found no difference in absorption of calcium and phosphorus between control and nematode-infected lambs. Assuming that the ash weight of bone is entirely Ca (PO ) 3 4 2 the calcium:phosphorus ratio is ideally 1.93 (Personal com-munication with Dr. H. Copp, Faculty of Medicine, Head, Department of Physiology, University of Br i t i s h Columbia.). Inspection of the results of femur analyses presented in Table VII shows that values both higher and lower than the ideal ratio were obtained within both the infected and the con-tr o l groups. There seems to be no evidence that the calcium: phosphorus ratio in the grouse femur is affected by D. nasuta infections. It was suggested by Dr. Copp that, i f the mineral compos-ition of the bones does not vary significantly, bone breakages may be due to malfunction in the formation of collagen (protein anabolism). The deceleration of feather production is not precipitous since feather checking f a i l s to appear. It might be postulated that a form of economization is present in young chicks during the early weeks of infection. The feathers of greatest sur-vival value are produced at the appropriate time, but perhaps at the expense of those of less significance. These results could be explained by a disturbance of the normal protein metabolism of the bird. - 76 -Section V IMMUNOLOGY AND RESISTANCE It was possible to examine, experimentally, several of the factors which may be responsible for the low incidence of infection in yearling birds (4$) and the absence of these Infections from adult birds under natural conditions. Reports on these experiments which were designed to illustrate (a) the presence of circulating antibodies for D. nasuta, (b) "the presence of age resistance to infection and (c) the effect of reinfection, are presented here. The experimental infections of the domestic fowl, which illustrates a different type of resistance to D. nasuta infections, may be found in Appendix C. Immunology Test for perilarval precipitates Third stage larvae were incubated at 39° C. in heparinized plasma from infected hosts. Three plasma donors which had been infected for 3, 5 and 8 months respectively were used. The larvae were examined frequently for the formation of precipitates around the mouth, excretory pore and anus. Larvae were also incubated in whole blood from non-infected birds to determine i f the larval sec-retions would lyse or coagulate the red blood c e l l s . The res-ults of these experiments are presented in Table VIII. Modified Ouchterlony gel diffusion test Plasma from the 5 and 8 month donors mentioned above and from non-Infected controls were used in conjunct ion with the two antigens prepared from larval and adult worms. No precipitates were obtained on - 77 -any of the plates. A repetition of the test, using the 8 month donor only, again failed to produce precipitates. Intradermal test Larval antigen, adult antigen and saline controls were injected in three intradermal sites on infected and control grouse. One infected bird (8 months), one recovered bird (26 months) and one control bird were sub-mitted to the tests. Sites of injection were observed every 5 minutes for the f i r s t 15 minutes, every half hour for the f i r s t 2 hours and thereafter once a day for 5 days. No reaction was observed except an immediate slight erythema probably due to the i r r i t a t i o n of injection since i t also occurred at the sites of saline injection. Reinfection Test One adult blue grouse hen, i n i t i a l l y infected 26 months prior to the reinfection tests, was ut i l i z e d . The bird had failed to produce D. nasuta ova in fecal samples for at least 12 months prior to these tests. Given 20 larvae, the hen ceased to eat three days after the administration and died on the ninth day. Autopsy showed a complete proventricular intussusception. The proventricular mucosa was hardened, black and necrotic. Age Resistance Attempts to infect blue grouse at 5 to 31 months of age were a l l successful. Both males and females infected 4 to 5 months before the mating season participated fully in the breeding activity. Subsequent autopsy of 3 laying females and - 78 -Table VIII Results of incubation of third stage larvae of D. nasuta in a variety of media. Medium No. of used larvae Duration (days) Results Physiological saline 15 5 no precipitates Plasma from controls 15 3- 5 no precipitates Plasma from infected birds 28 3- 5 no precipitates Whole blood from controls 12 3- 5 clumping of red blood cells - 79 -one displaying male yielded D. nasuta in each case. Discussion The low prevalence of natural infections of D. nasuta found in yearling and adult blue grouse compared to chicks can be explained by several hypotheses based on two alternate assumptions, (a) spontaneous natural healing occurs during the winter months (b) the majority of infected birds do not sur-vive the winter. Both (a) and (b) may operate simultaneously but for the sake of convenience, they w i l l be considered separately. (a) Spontaneous natural healing would occur i f one or more of the following hypotheses were true. (1) the l i f e span of D. nasuta adults is of short duration (2) the host develops an active immunity against the invader (3) physiological maturity of the host provides an unsatisfactory substrate for the parasite (age resistance) . (4 ) the winter diet (conifer foliage) contains anthelminthic agents (b) The survival of infected birds over the winter months would be low (Bendell, 1955) i f we assume that:-(1) P. nasuta has a debilitating effect on i t s host (2) winter conditions place a greater demand on the survival capabilities of the bird than do those of the summer - 8 0 -Experimental results have shown that the l i f e span of the nematode in laboratory hosts is 9 months to one year. If chicks in the f i e l d become infected soon after hatching in mid-June, the infection should s t i l l be present in the spring and early summer of the following year. Exploration of the other pos s i b i l i t i e s showed that there was no evidence that age related physiological changes or sexual maturity of grouse had any effect on the a b i l i t y of D. nasuta to establish new infections or to remain in old infections, no evidence of c i r -culating antibodies for D. nasuta, and no evidence that douglas f i r boughs, used to supplement the winter diet of the experi-mental birds, had an anthelmintblc effect. Therefore, a much higher prevalence of infection could be expeoted among yearling and adult grouse. Since v this is not the case, i t must be assumed that the hosts have been unable to survive the winter. It has been proved that this nematode has a seriously d e b i l i t -ating effect on i t s hosts. Such infected birds are more like l y to succumb to the additional stresses of f a l l migration and winter maintenance. Only those birds with light infections (e.g. less than 10 worms) would then survive. This would, in part, explain the small numbers of worms found in the yearling birds examined by Bendell (1955). Reinfection of previously infected birds seems unlikely (see below). The infection of yearlings and adults which have not been previously exposed to D. nasuta is possible because of the absence of age resistence. Apart from accidental contacts, however, this is equally unlikely. Examination of the crop contents of mature birds (personal observation and - 81 -communication with Bendell) indicated that animal matter is rarely present. Mature birds are effectively isolated from infection by the change from a carnivorous diet to an herb-ivorous one. That D. nasuta larvae do not invade and migrate through the tissues of the host would in part explain the absence of specific circulating antibodies. However, the extensive local host reaction m the form of inflammation, cellular i n f i l t r a -tion and mucosal hyperplasia; and the more generalized mani-festations of red c e l l loss (probably from agglutination) and anemia suggest that the worm would induce antibody formation. The non-discovery of antibody does not, of course, preclude it s presence especially since only aqueous extracts of worms were uti l i z e d . However, assuming that the glandular secretions of the third stage larvae are responsible for the loss of erythrocytes, the test for perilarval precipitates should be positive. This test, however, was repeatedly negative. The nature of the lesion and its similarity to the human nasal polyps formed in response to an allergen suggested to Dr. H. E. Taylor (personal communication), Head, Department of Pathology, University of Brit i s h Columbia, a reaction of the delayed hypersensitivity type. Circulating antibodies are not required for this reaction. Skin tests should be positive but delayed. It is possible that the aqueous extracts of larvae and adults did not contain the appropriate antigenic complex and hence the intradermal tests werfe negative. The fulminating reaction of the proventriculus to rein-fection with D. nasuta may indicate sensitization of the - 82 -tissues by the earlier infection. If this is the case, i t lends additional support to the delayed hypersensitivity hypothesis. Additional data concerning this aspect of the disease are needed. - 83 -Section VI APPEARANCE, BEHAVIOUR AND SURVIVAL OP INFECTED CHICKS Appearance and Behaviour Infected birds usually remained as alert and active as the controls. There were, however, several exceptions. Two chicks, infected at 8 days of age with 22 and 16 worms were less active than the controls. They frequently sat with feathers ruffled, gradually became lame, and, eventually, completely inactive except for feeding. Loss of pigment in the eye (Cram, 1928) was noticed only in these two birds. Death ensued 2-g- and 3i months after i n i t i a l infection. These two birds ate well, but remained small and emaciated, and feathered out slowly and incompletely. A ruffed grouse chick infected with 11 worms at 10 days of age was much less active than i t s brood-mates, frequently sitting with ruffled feathers. Although no signs of lameness developed, this chick was vis-ibly and measurably (Fig. 29) smaller than the ruffed grouse chicks of the same brood. Three days after i n i t i a l infection chicks up to 4- weeks of age passed bubbly brown droppings. After 3 to ? days, the droppings resumed their normal appearance. Two female chicks infected with 16 and 10 larvae at 5 months of age showed no signs of the disease other than re-current anorexia. Approximately every other month over a period of two years, these birds fasted for up to 10 days (determined by lack of feces). They remained healthy, however, coming into breeding condition and producing eggs. - 84 -Survival The p r i n c i p l e factor which controlled the scope of t h i s study was the numbers of 3rd stage larvae which were available for i n f e c t i n g chicks. Experiments were designed to get the maximum amount of information (on blood and growth) from as many infected chicks as possible. It was not des-'irable, for t h i s reason, to give the birds i n f e c t i o n levels which would have proved f a t a l before information of the course of the disease could be obtained. I n s u f f i c i e n t larvae and chicks were available to carry out experiments s p e c i f i c a l l y designed to show the i n f e c t i o n l e v e l s required to produce death of the infected hosts at various ages. Nevertheless, some of the infections did prove f a t a l . Two birds mentioned above, and the grouse used i n the r e i n -f e c t i o n experiments died as a d i r e c t result of the i n f e c t i o n . Pour infected yearlings and adults died with varying degrees of proventricular intussusception a f t e r they had been a c c i d -e n t a l l y frightened. The l e v e l s of i n f e c t i o n i n these birds were not s u f f i c i e n t to produce disease signs or cause death. Probably the sudden shock caused a violent contraction of the proventriculus which telescoped because i t s walls were weakened by the i n f e c t i o n . In addition an undetermined number of infected and control birds died of gizzard erosion. Since the records of deaths of control birds are incomplete, i t i s d i f f i c u l t to say whether the incidence of deaths from gizzard erosion was higher i n infected birds although t h i s appeared to be the case. The two female chicks mentioned above died, ultimately, from gizzard - 85 -erosion and i t is suspected that their repeated anorexia was an expression of this malady. GENERAL DISCUSSION The host-parasite relationship,inasmuch as i t involves an intimate interaction between two organisms, offers problems of great complexity to the investigator. Probably both the parasite and i t s host are affected or changed by this assoc-iation. It i s , however, d i f f i c u l t to ascertain the effects of the host on the parasite and this study is concerned only with the effect of the parasite on i t s host. Several of the types of injury which may be inflicted on a host by a parasite are illustrated by the relationship be-tween D. nasuta and the blue grouse. Studied specifically in this respect, were the injurious effects of this helminth on the proventriculus, blood, growth and development of the i n -fected host. The Proventricular Lesion The form of the lesion in the blue grouse corresponds most closely to that described for D. nasuta infected pigeons by Wasielewski and Wulker (1919) and Hwang et a l (1961). The conflicting interpretations of the tissue changes given for the pigeon by Cram (1928), for birds by Wehr (1948) and for the ruffed grouse by Edminster (I947) may be due, in part, to cursory examination, partial autolysis of tissues or differing responses to infection on the part of different host species. - 86 -This specific response is perhaps best illustrated by the domestic fowl where different strains showed variation in susceptibility to p. nasuta in this study and in the studies by Cram (1931) and to Asoaridia lineata (Ackert et a l , 1934). In this respect, i t was also noted that small hemorrhages were more numerous in the lesions of ruffed grouse than of blue grouse. In the blue grouse, the tissue changes observed in the papillomas are consistent regardless of the age of the host or the number and sex of the worms present. Increase in the number of nematodes only serves to increase the severity and spatial extent of the lesion. The gradual migration of adult p. nasuta outwards from regions of greatest papilloma devel-opment to the periphery of the lesion further extends the dimensions of this structure. The size of the lesion, then, is not only a function of the number of infecting nematodes but also of the length of time these worms are present. Several hypothesis- can be presented to explain this ex-tensive reaction of the host tissues. Undoubtedly the pres-ence of the worms is mechanically i r r i t a t i n g and produces a chronic inflammation which may in time result in hyperplasia of the proventricular tissues. However, i t is possible to cite examples of nematodes (Tetrameres sp., Rusguniella sp., Bchinurla sp.) which attack the proventriculus of birds and cause mechanical i r r i t a t i o n without producing hyperplasia of the tissues. The secretion by the worms of a tissue growth factor may also be postulated. However, a more tenable hypothesis is - 8? -suggested by the similarity in structure between the prov-entricular lesion and nasal and colonic "inflammatory" polyps in man. Tt is generally accepted that mucous nasal polyps are initiated by an allergic inflammatory response, and i t is entirely possible that the papillomatous growth in the proventriculus of the blue grouse is an allergic res-ponse to D. nasuta. The formation of this lesion is highly significant to the health of the host. It may form an effective obstruction to the immediately subtending deep glands and also tends to narrow or occlude the lumen of the proventriculus. Enlarge-ment of this organ to accommodate the proliferative growth results in hypertrophy and weakening of the muscular walls and death from intussusception may result. The chronic inflammation increases the production of mucous from the proliferated muc-osal epithelium and there may be alteration of the secretion of digestive juices from the deep glands. Both of these factors may alter the digestive capabilities of the infected birds. Although the optimal pH for avian pepsin and trypsin digestion, and the relative Importance of each enzyme for protein digestion in the bird are unknown, (Sturkie, 1954) i t is possible to postulate that alteration of the secretion of the proventricular digestive juices (pepsin and HCL) may change the pH of the digestive tract and hence the digestive and assimilative capacities of the infected host. Growth and Development of the Infected Host Three aspects of growth and development in the blue grouse - 88 -were considered (a) weight gain (b) calcium and phosphorus content of bones and (c) feather development. Whether or not the growth and development of Infected birds is impaired i s dependent on the number of nematodes present and the age at which they are acquired. Infections of less than 10 nematodes did not affect the growth or dev-elopment of chicks older than 8 days regardless of when they were acquired. Infections of 11 to 20 worms did not apprec-iably affect the growth of chicks infected at 11 weeks of age (after the rapid growth phase). Birds which have achieved this state of maturity are probably less vulnerable. Their growth Is slower and feathering and bone development are a l -most complete. Therefore, infections of several hundred worms may be necessary to produce demonstrable changes in their growth. The weight gain of chicks infected with more than 10 worms between 1 and 4 weeks of age was impaired although more worms were required to retard the growth of chicks infected at 4 weeks than were required at 1 week. There are two phases "in the growth of infected birds when the growth may be severely retarded or loss of weight is ex-perienced. These phases correspond approximately in time to the two c r i t i c a l periods in the hematology of infected birds (when erythrocyte levels are at a minimum) and probably re-fle c t the general debility of the host at these times. No evidence was found that the calcium:phosphorus ratio in the femur of infected birds was disturbed although these bones were frequently bowed or broken. An insufficient number of femurs was analysed to definitely conclude that the - 89 -mineral metabolism is not affected. However, the weakness of these bones may not be an indication of mineral deficiency or imbalance but rather of malfunction in the formation of the collagen matrix of the bone. Retardation of feather development appeared only in birds infected with more than 10 worms before the age of 2 weeks and affected only the lesser body coverts which appear after 3 weeks of age. The impaired growth and development of infected birds cannot be attributed to reduced food intake. Experiments have shown that infected hosts eat as much as the controls and that they require a greater quantity of food to produce a unit increase in body weight. It appears that infected birds are unable to u t i l i z e the ingested food as efficiently as the controls. The results of the experiments on weight gain, mineral content of bone and feather development a l l suggest that pro-tein digestion and utilization are affected. The significance of the proventricular lesion to protein digestion in the host has already been discussed (see above). A second,or concom-itant possibility is the inhibition of the enzyme systems of the host by antienzymic secretions of the nematode. An anti-enzyme which inhibits pepsin and trypsin digestion has been reported from Asoaris lumbrlcoides by Sang (1938). The sec-retion by D. nasuta of an antienzyme is hypothetical. However, there is adequate evidence of disturbance of the protein met-abolism of the host and further research is required to det-ermine how this effect is accomplished by the parasite. - 90 -Hematology of the Infected Host The two fluctuations in the numbers of hemocytes illustrate the fact that there are two c r i t i c a l periods in the course of the infection. These periods, characterized by anemia and leucocytopenia occur approximately 2 days and 25 days after i n i t i a l infection. The f i r s t c r i t i c a l period is possibly caused by the secretion of a harmful substance by the invading larvae. This substance may be correlated with the loss of a glandular material from the buccal region of 3rd stage larvae during the f i r s t 3 days of Infection. The subsequent rapid recovery of blood elements substantiates this view. The presence of a larval secretion i s hypothetical. However, the disappearance of the glandular substance from the invading larvae and the appearance of agglutinated erythrocytes in the circulating blood are highly suggestive. Such a sec-retion may alter the proventricular tissues and assist in the establishment of the larvae. Agglutination of erythrocytes may be a direct effect of the secretion or, indirectly, i t may alter the erythrocytes so that they are agglutinated by the host. The second c r i t i c a l period seems to be a response to the maturation of the worms. The onset of the decline in hemo-cytes may be directly correlated with the occurrence of the moult from 4th stage larvae to adult. It is possible that the release of a harmful substance during the moult would explain this precipitous decline in blood c e l l numbers, although the moult from 3rd stage to 4th stage did not produce a similar - 91 -result. Soulsby (1961) has shown that the exsheathing fluid released by Hemonohus oontortus in sheep during moult is strongly antigenic and stimulates the host to produce antibodies. He has also shown that each larval and adult stage w i l l stimulate the production of antibodies which are specific for the stage involved. It is possible that the exsheathing f l u i d of the 4th stage larvae of p. nasuta is particularly i r r i t a t i n g to the host tissues and is responsible for the general somatic disturbances (growth and hematology) observed 3 weeks after infection. It may also be postulated that increased activity of the nematodes at this time as they migrate through the tissues of the lesion seeking out mates, with resultant focal accumulations of larvae, cause the in-creased inflammation observed. The i r r i t a t i o n of the worms in the inflamed' edematous tissues may precipitate the onset of the local hemorrhages which, in turn, are considered res-ponsible for the anemia manifested. These changes in the blood elements occur regardless of the number of nematodes present or the age of the host. In-crease in the numbers of D. nasuta serve to accentuate the amplitude of the fluctuations - particularly that of the second c r i t i c a l period. This low level of circulating hemo-cytes corresponds in time with the maturation of the worm, the appearance of hemorrhages in the lesion, the elongation of the individual papillomatous elements of the lesion and the severely retarded phase of weight gain. - 92 -Immunology and Resistance of Infected Birds Yearling and adult birds were susceptible to infections of D. nasuta. There i s , then, no evidence of age resistance. Also, breeding birds did not lose their infections showing that the sexual hormones of the host did not have a detrim-ental effect on the parasite. The absence of the parasite from adult blue grouse in the f i e l d can only be explained by the fact that the feeding habits of these birds, which sub-sist mainly on vegetable matter, effectively isolate them from acquiring the infection. The results of the immunological experiments are by no means conclusive. It can be said, however, that the tech-niques used failed to illustrate the presence of circulating-antibodies ^ for p. nasuta. The absence of precipitates around the orifices of larvae incubated in plasma from infected birds is probably most significant in this respect. It is suggested that the papillomatous lesion of the proventriculus may form in response to an allergen and that the reaction may be of the delayed hypersensitivity type. Circulating antibodies are not demonstrable in this type of sensitivity. The similarities, as previously mentioned, between the structure of the proventricular lesion and of the nasal and colonic polyps formed in man in response to an allergen help to substantiate this view. In one instance only was there an opportunity to study the reinfection of a bird. The fulminating character of the reaction as compared to a primary infection was so marked that i t might be considered a secondary response in an already sensitized bird. - 93 -It appears that the pathogenicity of D. nasuta in the "blue grouse cannot be attributed, to a single type of damage but rather to a complex of injurious effects. Both mech-anical and physiological effects have been illustrated. There is no evidence, as yet, that the nematodes u t i l i z e and hence deprive the host of essential nutriments or elaborated products such as blood. There is no doubt that D. nasuta Infections are d e b i l i t -ating to the host and i t has been demonstrated that they can prove f a t a l . The desire to investigate the disease process at length and the lack of sufficient numbers of larvae res-tricted the number of fatal infections in this series of experiment s. It is unwise to apply the results of laboratory findings directly to the interpretation of f i e l d results. It is un-li k e l y , for example, that an animal confined in a laboratory itfill react, biologically, as i t would in i t s natural environ-ment. However, bearing this fact in mind, and having a basic knowledge, gained from laboratory research, of the biological processes involved, i t is possible to speculate on the causes of certain phenomena observed in the f i e l d . The f i e l d obser-vations considered here are tho'se of Bendell (1955) who sug-gested that D. nasuta in conjunction with Plagiorhynchus  formosus were causal agents of the extreme mortality which he recorded for blue grouse chicks on Vancouver Island, B r i t i s h Columbia. A l l experimentation was performed in the laboratory on cage confined blue grouse chicks. These birds had ready - 9k -access to food, water and sources of heat and gained weight more rapidly (over the period of experimentation) than wild chicks reported in the literature. They were exposed, however, to the adverse effects of confinement and lack of exercise, the only noticeable result of which was an elevated leucocyte count. The chicks in the f i e l d are exposed to continuous acquisition of D. nasuta over the summer months (several stages, larvae and adults, may be found in the proventriculus at one time), to parasitism by organisms other than p. nasuta and to the stress of environmental factors. From the laboratory studies i t is evident that the effects of D. nasuta on the blood elements and probably on the protein digestion of the host pose the most serious threat to survival. Of these two, the effect on the blood is probably of greater significance because of the sudden c r i t i c a l lows which the various elements assume at certain stages of the infection. It is impossible to interpret the hematological evidence with-out a knowledge of when and how an infection was acquired. This, coupled with the lack of information concerning the ef-fects on the blood elements of other parasites harboured by the host, rendered the study of the blood of f i e l d infected birds unproductive. The effect of continuous acquisition of worms is not known. However, from the laboratory results, a constant depression of the numbers of both red and white cells could be expected. The presence of these worms is debilitating and lowered resistance probably predisposes the wild host to suffer more - 95 -severely from the adverse effects of inclement weather and other parasite burdens. Chicks infected under 2 weeks of age would be most likely to succumb from even light infections under these conditions. Particularly i f they suffer from anemia immediately after acquisition of the worms as do the experimental chicks. Essentially, the laboratory experiments substantiate the conclusions which Bendell (1955) drew from his f i e l d observa-tions. The results of further investigations by Bendell on blue grouse populations (as yet umpublished) may reveal that D. nasuta is not so prevalent in the study area as i t was in the years preceedmg 1955 and that factors important in the control of these populations are operative without the presence of damaging parasites. To summarize, p. nasuta is in t r i n s i c a l l y capable of producing a fatal infection. Whether or not a natural infec-tion is fatal depends on the number of worms present and the age of the host when they are acquired, on the rate at which they are acquired, on the health of the host, i t s parasite burden, and the environmental stresses to which i t is exposed. Consequently, i t can be seen that no fixed number of nematodes can be used as a criterion for an infection level which w i l l prove f a t a l . Bendell (1955) found a low incidence of D. nasuta infec-tions in yearling blue grouse and the absence of this parasite in adult grouse. Experiments have shown that the nematode lives 9 to 12 months in the captive bird, that there is no age immunity to this infection and that birds which have recovered - 96 -from their f i r s t infection may be sensitive to reinfection. The absence of D. nasuta infections in adult birds can be explained by their food habits which effectively isolate them from acquiring an infection. If, however, an adult which had been infected as a chick and recovered, accidently acquired D. nasuta larvae, i t would probably succumb to the hypersensitive reaction. It is probable that Infected chicks suffer greater mortality under conditions of winter stress and that infected yearlings represent those chicks with light infections which were able to survive the winter. - 97 -SUMMARY AND CONCLUSIONS (1) This study was conducted to determine the effects of infections of Dispharynx nasuta on two subspecies of blue grouse chicks, Dendragapus obscurus fuliginosus and D. o. pallidus in B r i t i s h Columbia. Various aspects of the disease condition such as development of the lesion, effect on growth, development, hematology, survival, immunology of the host, and development and ecology of the parasite were examined. (2 ) Blue grouse chicks of several age groups, maintained in the laboratory, were infected experimentally with varying numbers of D. nasuta larvae. (3) Both control and infected chicks were weighed and their blood sampled regularly. Measurements were also made of the food intake, feather production, and calcium and phosphorus content of bones of the chicks. Proventriculi of experiment-al l y infected hosts were collected at selected intervals after i n i t i a l infection and examined histologically. (4) Various immunological procedures were used in an attempt to discover circulating antibodies for D. nasuta in infected chicks. (5) It was found that, in general, the severity of the disease process is directly correlated with the number of worms present and the age at which the infection is acquired. Older birds are more tolerant of heavy infections. (6) The lesion is a papillomatous outgrowth of the super-f i c i a l mucosa of the proventriculus. The deep glands of the mucosa may exhibit papillomatous changes of the epithelium - 98 -lining the main secretory ducts and may, in time, atrophy from the effects of pressure and occlusion. Papillomatous alteration of the squamous epithelium of the posterior end of the esophagus may occur with cystic changes of the mucous glands in this region. The koilin of the interventriculus is softened and elongated and the secreted strands tend to remain separate and distinct. There is no evidence of necrosis in any of these tissues. (7) The lesion in the i n i t i a l stages of infection shows edema of the plicae of the mucosa with massive cellular i n f i l -tration. The cellular exudate, composed mainly of lymphocytes with a few monocytes and granulocytes reaches peak development between the 2nd and 3rd. weeks of the infection. Thereafter, the fibroblasts cause a fibrosis of the plicae, which forms the connective tissue stroma of the papilloma. By 3*2 weeks after infection, the papilloma seems to have reached i t s max-imum development in size. There is a gradual Increase in the numbers of granulocytes found m the tissues of the lesion and at 12 weeks they are characteristically found in large numbers throughout the papilloma. Small local hemorrhages form in these tissues approximately 3 weeks after i n i t i a l infection. (8) The hematology of infected chicks is characterized by dramatic fluctuations in the numbers of a l l hematocytes. There are, during the i n i t i a l stages of the disease, two c r i t -i c a l periods when the numbers of a l l blood cells are low. It is probable that a secretion of the invading larvae which causes acute inflammation and agglutination of the erythrocytes is responsible for the f i r s t decline. The second decline, - 99 -occurring between 2 and 3 weeks after infection may be the result of maturation of the worms. The onset of this decline coincides with the f i n a l larval moult. It is sug-gested that the host may react to some component of the ex-sheathing f l u i d . As well, increased activity and concentration of the nematodes in foci may, by i r r i t a t i o n , effect a second period of acute inflammation with consequent concentration of leucocytes in the tissues and initiate small hemorrhages in the lesion. After k to 5 weeks, the numbers of hematocytes tend to return to normal. There i s , however, evidence of chronic an-emia and leucocytosis. The latter is characterized by heter-ophilia, eosinophilopenia, lymphocytosis of small forms and lymphopenia of the larger forms. (9) The growth of chicks infected with less than 10 worms is not noticeably affected. However, the growth of those infected with more than 10 worms before 2 weeks of age is slower than that of the control chicks 3 weeks after administration of the larvae. This retardation of growth is not due to anorexia since the studies of the food intake of infected birds have shown that they eat as much as or more than the controls. It is postulated that the lesion alters the digestive and assim-il a t i v e functions of infected chicks. (10) No correlation could be found between the calcium:phos-phorus ratio in bones and the presence of the infection. Bone breakages exhibited in infected chicks may then be the result of altered protein metabolism. (11) There is no evidence that the growth of primaries, - 100 -secondaries, retrices and main body coverts is affected. There i s , as well, no evidence of feather checking. However, the growth of feathers in the more protected regions (those which appear from 3 to 5 weeks of age) may be severely re-tarded and they may not appear by 12 weeks of age. This is interpreted as a form of economization. The feathers of greater significance for survival appeared-to develop at the expense of those of lesser significance. These results also suggest impairment of the protein metabolism of the host. (12) No evidence of circulating antibodies was found with the techniques used. The papillomatous response of the provent-ricular tissue, the similarity of this reaction to the form-ation of the human nasal polyp, and the evidence of sensitiv-ity to reinfection a l l suggest that the tissue reaction may be the response to an allergen. The absence of circulating antibodies suggest a reaction of the delayed hypersensitivity type. (13) Infections of 16 and 22 worms administered at one week of age have proved fatal to laboratory raised birds. In this light, and considering the general debilitating effects of this nematode, the laboratory results suggest that the chances for survival of infected birds under natural conditions are poor. (14) The longevity of adult D. nasuta is approximately 9 to 12 months. The parasite probably survives during the winter months as a larva in the intermediate host, as an egg in grouse feces, and to a lesser extent as an adult in the blue grouse. - 101 -(15) Additional information on the l i f e cycle of D. nasuta with descriptions and durations of the larval forms which occur in the definitive host are included in this study. (16) Oniscus asellus is reported as a new intermediate host for this nematode. - 102 -LITERATURE CITED Ackert, J. E., Eisenbrandt, L. L., Glading, B. and Wilmotb, J. H., 1934. On the comparative resistance of six breeds of chickens to the nematode Ascarldia lineata (Schneider). J. Parasitol., 20, 127. Ackert, J. E. and Wilmoth, J. H., 193^. Resistant and sus-ceptible strains of white minorca chickens to the nema-tode, Ascaridia lineata (Schneider). J. Parasitol., 20, 323-324. Alicata, J. E. , 1938. Studies on poultry parasites. Hawaii Agr. Expt. Sta. Rep., 1937, 93-96. Allen, A. A., 1925. The grouse disease in 1924. Bull. Am. Game Protect. Assn., 14, 11, 12, 20. Allen, A. A. and Gross, A. 0., 1926. Ruffed grouse Investi-gation, season of 1925-26. Amer. Game. J L 5 , 81-86. Andrews, J. S., 1939. Effect of infestation with the nema-tode, Cooperia curticei, on the nutrition of lambs. J. Agric. Res. , _£Z, 349-362. Andrews, J. S., Kauffmann, W. and Davis, R. E., 1944. Effects of the intestinal nematode, Trichostrongylus colubriformis. on the nutrition of lambs. Amer. J. Vet. Res., j £ , 22-29. Bendell; J. F., 1955* Disease as a control of a population of blue grouse, Dendragapus obscurus fuliginosus (Ridgway). Can. J. Zool., 21, 195-223. Bendell, J. F., 1955a. Age, molt and weight characteristics of blue grouse. Condor 354-361. Biely, J. and Palmer, E. I., 1935. Studies of total erythro-cyte and leucocyte counts of fowls. III. Variation in number of blood cells in normal fowl. Can. J. Res. Sect. D. Zool. Sci., 12, 61-71. Blain, D. A., 1928. A direct method for making total white blood c e l l counts on avian blood. Proc. Soc. Expt. Biol. Med., 25, 594-596. Boddie, G. F., 1956. Diagnostic Methods in Veterinary Medicine. 4 t h Ed. J. B. Lippincott Co., Philadelphia. Bump, G., Darrow, R. ¥., Edminster, F. C. and Crissey, W. F., I947. The ruffed grouse, l i f e history, propagation, management. New York State Cons. Dept. - 103 -Calhoun, M. L., 1954. Microscopic anatomy of the digestive system of the chicken. Iowa State Coll. Press, Ames, Iowa. Chabaud, A. G. and Petter, A. J., 1959. Essal de c l a s s i f i c -ation des nematodes Acuariidae. Ann. de Parasitol. jS4, 322-330. Clarke, E. P. and Collip, J. B., 1925. A study of the Tisdall method for the determination of blood serum calcium with a suggested modification. J. Biol. Chem., 6 ,^ 461-464. Cook, S. P., 1937. A study of the blood picture of poultry and i t s diagnostic significance. Poultry Sci. 16, 291-296. Cook, S. P. and Scott, K. G., 1935a. Apparent intoxication in poultry due to nitrogenous bases. Science 82, 465-467. Cook, S. P. and Scott, K. G., 1935b. A bioassay of certain protein supplements when fed to baby chicks. Proc. Soc. Expt. Biol. Med., _3_3_, 167-170. Cram, E. B., 1928. Nematodes of pathological significance found in some economically important birds in North America, U. S. Dept. Agr. Tech. Bull., 49. Cram, E, G., 1930. Pathological conditions ascribed to nematodes in poultry. U. S. Dept. Agr. C i r c , 126. Cram, E. B., 1931. Developmental stages of some nematodes of the Spiruroidea parasitic in poultry and game birds. U. S. Dept. Agr. Tech. Bull., 227. Cram, E. B., 1932. Additional observations on bird hosts of Pispharynx spiralis. J. Parasitol., 18, 310. Edminster, P. C , 1947. The Ruffed Grouse. MacMillan Co., New York. Pantham, H. B., 1910. Observations on the blood of grouse. Proc. Zool. Soc. London. 722-731. Porkner, C. E. , 1928. Blood, and bone marrow cells of the domestic fowl. J. Exp. Med., 50, 121-141. Franklin, M. C., Gordon, H. M., McLeod, H. and Macgregor, C. H., 1946. A study of nutritional and biochemical effects in sheep of infestation with Trichostrongylus colu-brlformis. J. Counc. Sci. Indust. Res.. 19^ 46-60. - 104 -Goblei, F. C. and Kutz, H. L. , 1945. The genus Dispharynx (Nematoda:Acuariidae) In galliform and passeriform birds. J. Parasitol., 31, 323-331. Gross, A. 0., 1925. Report on New England ruffed grouse investigation. Auk 42, 423-431. Holman, H. H., 1956. C l i n i c a l Haematology. Ch. XVIII in ' Diagnostic Methods in Veterinary Medicine Ed. by G. F, Boddie. 4th ed. J. B. Lippincott Co. Philadelphia. Hwang, J. C , Tolgay, N. , Shalkop, W. T. , Jaquette, D. S. , 19-61. Case report - Dispharynx nasuta causing severe proventriculitis in pigeons. Avian Diseases 60-65. Kramer, B. and Tisdall, F. F., 1921. A simple technique for the determination of calcium and magnesium in small amounts of serum. J. Biol. Chem., 42, 475-481. Lee, B., 1950. The Microtomist 1s Vade-Mecum. Ed. by J. B. G-atenby and H. W. Beams. 11th ed. The Blakiston Company. Philadelphia. Legros, C., 1864. Affection vermineuse insolite chez les gallinace's. Compt. Rend. Soc. Bi o l . , 218-219. Levine, P. P. and G'oble, F. C , 1947. in the Ruffed Grouse, Life History, Propagation, Management by Bump et a l . New York State Cons. Dept. Lucas, A. M. and Jamroz, C , 1961. Atlas of avian hematology. U. S. Dept. Agr. Monograph 25. Markell, E. K. and Voge, M., 1958. Diagnostic Medical Para-sitology. W. B. Saunders Co. Philadelphia & London. Olson, C., 1937. The normal variations of the cellular elements and hemoglobin in the blood of domestic chickens. Cornell Vet., 22, 235-263. Olson, C., 1948. Avian Hematology. Ch. 4 in Diseases of Poultry. Ed. by H. E. Biester and L. H. Schwarte. 2nd. ed. Iowa State Coll. Press, Ames Iowa. Olson, C. and Levine, P. P., 1939. A study of the cellular elements and hemoglobin in the blood of chickens experi-mentally infected with Capillaria columbae (Rud.) Poultry Sci., 18, 3-7. Osche, G., 1955. Bau, Entwicklung und systematische Bedeutung der Cordons der Acuariidae (Nematoda) am beispiel von Stammerinema soricis (Tiner, 1951) gen. nov. Z. f. Parasit., Bd. 12, 73-92. - 1 0 5 -Ouchterlony, 0 . , 1 9 4 8 . Antigen-antibody reactions in gel. Ark. f. Kemi. Miner. Geol., Bd 2 6 , No. 14. Palmer, E. I. and Biely, J., 1 9 3 5 . Studies of total ery-throcyte and leucocyte counts of fowls. IV Erythrocyte and leucocyte counts of birds raised in confinement. Can. J. Res. Sect. D., Zool. Sci., 13_, 85-88 Piana, G. P., 1 8 9 7 . Osservazioni sul dispharagus nasutus Rud. dei p o l l i e sulle larvae nematoelmintiche delle mosche e dei porcellioni. A t t i . Soc. I t a l . Sc. Nat. Milano, 2&, 2 3 9 - 2 6 2 . Roberts, J. C., i 9 6 0 . Ch. 7 in the Lymphocyte and Lymphocytic Tissue. Ed. by J. W. Rebuck. Int. Acad. Pathol. Monograph. Rogers, W. P., 1941. The metabolism of Trichinosed rats during the early phase of the disease. J. Helmin.thol. , 12, 87-104. Rogers, ¥. P. , 1 9 4 2 . The metabolism of Trichinosed rats during the intermediate phases of the disease. J. Helmin-thol., 2 0 , 1 3 9 - 1 5 8 . Sang, J. H., 1 9 3 8 . The antiproteolytic enzyme of Ascaris  lumbricoides Var. Suis. Parasitol., _3_0, 141-155. Schmeisser, H. C , 1 9 1 6 . Leukaemia of the fowl: spontaneous and experimental. Rep. Johns Hopkins Hospital. J _ 7 , 551-586. Shearer, G. D. and Stewart, J. , 1 9 3 3 . The effects of nema-tode infestations on the metabolism of the host. Part III Effects on mineral metabolism. Rep. Inst. Animal Pathol. Univ. Cambridge, j i , 87-94. Sobolev, A. A., 1 9 5 7 . Evaluation of the application of com-parative ontogenic method in the systematics of Spirurats (Nematoda, Spirurata) Zoologitcheski Journ., 36, 1 3 0 4 - 1 3 1 1 . Soulsby, E. J. L., I 9 6 I . Some aspect of the mechanism of immunity to helminths. J. Amer. Vet. Med. Assoc., 1 3 8 , 3 5 5 - 3 6 2 . Stiven, A. E., 1 9 5 9 . A study of the relationship between available food and nutritive requirements of blue grouse chicks. Unpublished. M.A. Thesis - University of British Columbia. Sturkie, P. D., 195^. Avian Physiology. Comstock Pub. Assoc. Ithaca, New York. - 106 -Taussky, H. H, and Shorr, E., 1953. A microcalorimetric method for the determination of inorganic phosphorus. J. Biol. Chem., 202, 675-685. Tiner, J. D., 1951. Dispharynx soricis n.sp. (Nematoda: Acuariidae) from the shrew Sorex obscurus alascensis, and associated host -oathology. Proc. Helm. Soc. Wash., 18, 64-70. Twisselman, N. M. , 1939. A study of c e l l content of the blood of normal chickens, with special reference to com-parative differential leucocyte counts made with supra-v i t a l and Wright's staining technics. Poultry Sci., 18, 151-159. Van Tyne, J. and Berger, A. J., 1959. Fundamentals of Orni-thology. J. Wiley and Sons, Inc., New York. Walford, L. A., 1946. A new graphic method of describing the growth of animals. B i o l / B u l l . , £0, 141-14?. 11 Wasielewski, T. von, and Wulker, G. , 1919. Zur Kenntnis der Dispharagus infection des Geflugelmagens und <fer dadurch bedingten geschwulstartigen Sehleimhautwucherungen. Zeit. f. Krebsforschung 16, 250-273. Wehr, E. E., 1948. Nematodes and acanthocephalids of poultry. Ch. 32 in Diseases of Poultry. Ed. by H. E. Biester and L. H. Schwarte, Iowa State College Press, Ames Iowa 2nd. ed. Wing, L., Beer, J., and Tidyman, W., 1944. Brood habits and growth of "blue grouse". Auk. 6 l , 426-440 - 107 -Appendix A. Determination of Age of Chicks Caught' in the Field The weights of field-caught chicks are unreliable indic-ators of age. There is frequently wide disparity in weight values between the chicks of a single brood. The extent of development of the feathers of chicks from a. brood is more consistent. The feathers are produced at a f a i r l y constant rate which may reflect their importance to the survival of the bird. Accordingly, the age of chicks caught in the f i e l d was determined by comparison of their feather development with that of control birds of known age. The following data were used as the aging c r i t e r i a . Designations for feather tracts and regions were taken from Van Tyne & Berger (1959). Feather Development of Control Chicks Age of chick (days) Development of feathers 1 primaries 5 to 6 mm. long secondaries 3 to 4 mm. long 3-4 scapulars break through periderm 5-6 primaries reach base of pygidium greater secondary coverts break through periderm 8-9 scapulars 25^ to 30 mm. long peridermal sheaths of retrices 5 to 7 mm. long peridermal sheaths present in sternal region of ventral tract 10-11 middle secondary coverts in periderm ear coverts in periderm sternal region of ventral tract breaking through periderm crural tract in periderm greater secondary coverts 10 mm. long - 108 -12-13 retrices break through periderm primaries and secondaries extend, just past end of pygidium scapulars continuous over back peridermal sheaths present in coronal region feathers breaking peridermal sheaths at base of neck (cervical region of spinal tract) feathers of abdominal region of ventral tract have broken through periderm no under wing coverts present 7 primaries present, 8th breaking through periderm 14-16 middle secondary coverts break through periderm 19-21 ear coverts break through periderm retrices 25 mm. long feathers of dorsal and pelvic regions, of spinal tract break through periderm 22-25 feathers of upper cervical region of spinal tract breaking through the periderm tract on tarso-metatarsus in peridermal sheaths or just breaking 30 ocular and l o r a l feather break through periderm 32 interramal feathers break through periderm Field-caught chicks ranged m age from 2 to 18 days with the majority ranging between 5 and 14 days. In using this technique of aging, the assumption was made that the feather development of f i e l d chicks is comparable to that of birds in the laboratory. There i s , so far, no evidence to the contrary. The greatest variation in feathering of the control chicks was found in the time of appearance of the retrices which broke through the q u i l l between the 9th and 12th days. No differences attributable to sex or subspecies could be - 109 -found in the times of appearance of the feathers. Sexing of the chicks of D. _o. fuliginosus was possible at 33 days. The ocular feathers which develop at this time are uniform in colour in the male and form a dark brown crescent under the eye. These feathers are speckled brown and white in the female. This differentiation is not possible for D. 0 . pallidus chicks and sexing of these birds can be accomplished shortly after 5 weeks by examination of the newly formed outer yearling retrices which are speckled in the female buti not (or less so) in the male. - 110 -Appendix B. Analysis of Logarithmic Transformation of Growth Data The volume of weight data collected for control birds warranted consideration beyond that which was required for the immediate purposes of this study. The Walford transfor-mation, based on weekly weight averages, omitted approximately one-fith of the available data. It also seemed advantageous" to determine the growth rate for each of the three phases of growth which became evident with a logarithmic transformation of both age and weight. The limits for the three phases of growth were selected by inspection of the log-log data plots and were as follows; 1 s t phase, male and female, 0 - l 4 days 2nd phase, male, 15-76 days female, 15-69 days 3 r d phase, male, 77-end days female, 70-end days The data were processed by an ALWAC dig i t a l computer and the resultant coefficients of regression are presented in Table IX. Table IX Computed Growth Rates for Blue Grouse Chicks of Two Sexes and Two Subspecies Raised in Captivity Phase fuliginosus fuliginosus pallidus pallidus males females males females 1 0 . 6 9 3 6 3 0 . 6 7 3 7 1 0 . 4 3 8 7 5 0.41211 2 O .60507 1 .51390 1 .71901 1 .56479 3 0.56139 0 . 5 5 3 2 1 O.5O897 - I l l -Appendix C. Experimental Infections of the Domestic Fowl with D. nasuta Infections of D. nasuta have been reported frequently from the domestic chickens (Goble and Kutz, 1945). An attempt was made to use chicks of Gallus domesticus for experimental infections during the winter months when blue grouse chicks were not available. Exper iment (1) Ten day-old Rhode Island Red cockerels were obtained from the Department of Poultry Science at the University of British Columbia. Of these, 7 were given from 25 to 125 larvae at 5 days of age and 3 were reserved as controls. Blood sampling commenced 5 days after administration of the larvae. No differences were observed between the blood samples of infected and control chicks. On autopsy, at 6 weeks of age, a l l chicks were negative for worms. As well, there was no evidence of tissue alterations in the proventriculus to indicate successful establishment of the larvae. Experiment (2) Two white Leghorn chicks were given D. nasuta larvae. One day-old chick received 8 larvae. On autopsy, 4 days later, the proventriculus was negative for p. nasuta. The mucosal surface of this organ, however, showed 3 patches of yellow, hardened, apparently necrotic tissue. Long strands of blood - 112 -stained raucous (positive Benzidine test) were associated with these areas. The deep glands subtending the yellow patches were also hemorrhagic. Samples of the intestinal contents taken at various levels from the duodenum to the rectum were a l l positive for blood. The second chick was given a single larva at 8 days of age. Sacrificed 24 hours later, the chick exhibited no hem-orrhagic areas in the proventriculus. However, long, thick strands of yellow mucous occupied an area 10 mm. wide by 7 mm. deep on the mucosa. Three regions of inflamed plicae x-jere present near the mouths of the deep glands. Adjacent to one of these inflamed areas, the third stage larva was found, i t s head embedded between the plicae and i t s body free in the lumen of the proventriculus. Cram (193D established experimentally the cross trans-mission, of D. nasuta from ruffed grouse to bobwhite quail and. the pigeon. She was, however, unable to established the cross transmission of this worm from the ruffed grouse to domestic chicks and postulated that the chicken from long association may have become much more resistant to infection with this parasite than are game birds. Variation in susceptibility of various strains of chickens to infections of Ascaridia  lineata has been reported by Ackert et a l (1934) and Ackert and Wilmoth (1934). It is evident in this study, that attempts to transmit D. nasuta from blue grouse to chicken were unsuccessful. (No d i f f i c u l t i e s were encountered in the transmission of this - 113 -worm from blue to ruffed grouse.) The tissues of the chicken proventriculus respond acutely to the i n i t i a l invasion of D. nasuta, larvae. It appears that this response is sufficiently intense to render the proventriculus inhospitable to the establishment of the parasite. Reactions of this severity were never encountered in the blue grouse proventriculus. It is possible that physiological races of p. nasuta or, more likely, genetic strains of chickens of varying susceptibilities have developed. The acute reaction found in the proventriculus is suggestive that the tissues of the strain of chicken used were highly sensitive to the secretions or excretions or both of D. nasuta. 

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