Robert M. Stabler

EFFECT OF CERTAIN LABORATORY PROCEDURES ON VIRULENCE OF THE JONES' BARN STRAIN OF TRICHOMONAS GALLINAE FOR PIGEONS.

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

Hematozoa from Colorado Birds. II. Falconiformes and Strigiformes.

Blood smears from Colorado Falconiformes (186 birds of 14 species) and Strigiformes (36 birds of 9 species) were examined for hematozoa. Where available, bone marrow was checked for trypanosomes. Ninety (48%) of the Falconiformes and 25 (69%) of the Strigiformes showed some parasitemia. New host-parasite associations were: Haemoproteus-goshawk, ferruginous hawk, barn owl, flammulated owl; Leucocytozoon-Coopers hawk, rough-legged hawk, ferruginous hawk, marsh hawk, sparrow hawk, barn owl, flammulated owl, short-eared owl; Trypanosoma-goshawk, sharp-shinned hawk, pigeon hawk, sparrow hawk, barn owl, flammulated owl, pigmy owl, long-eared owl; Hepatozoon-red-tailed hawk; Microfilaria-goshawk, sparrow hawk, long-eared owl. The ferruginous hawk, prairie falcon, pigeon hawk, flammulated owl, and pigmy owl appear to have been examined for the first time. Plasmodium hexameriumn is reported for the second time from the screech owl (first described as P. oti by Wolfson, 1936). Reporting on a survey of the hematozoa from Colorado birds, Stabler and Holt (1963) presented data from 464 members of the Columbiformes. The present paper deals with members of the Falconiformes and Strigiformes. Data from members of other orders will be published as the slides are examined. The birds were captured or shot within the state of Colorado. Live birds were bled from Received for publication 16 August 1965. a snipped toenail, dead ones from the thoracic cavity. Tibial marrow was examined in a few cases. Films were examined following staining in Giemsa. The bone marrow was examined fresh in physiological saline. The Falconiformes were represented by 186 birds of 14 species, the Strigiformes by 36 birds of 9

Studies of the age and seasonal variations in the blood and bone marrow parasites of a series of black-billed magpies.

Reports of the blood parasites of birds are extensive. According to Manwell (1955a), however, there have been very few attempts to study the relative incidence of iialaria and other protozoan infections of the blood in juveniles, or to discover seasonal variations in the incidence of such infections. Herman (1938) reported on the incidence of Haemoproteus in some juvenile and adult chipping sparrows. Manwell (1955b) compared the blood parasites of juvenile and adult robins from New York State and the high Rockies. Coatney and Jellison (1940), Wagner (1946), and Hagen and Olsen (1957), all reported on the hematozoa of black-billed magpies of various age levels. None of these authors, however, too clearly defined the age implications of such terms as nestling, fledgling, juvenile, iimmature, and adult. In a very extensive study of the plasmodia of Kern Co., Calif., birds, Herman et al (1954) noted the organisms in three welldefined age groups of 3 species of birds. Similar observations on the parasites of Canada geese in specific age categories were made by Levine and Hanson (1953), Wehr and Herman (1954), and Hanson et al (1956). Even less has been published on seasonal variations in parasite incidence. Jordan (1943) noted some seasonal influence on Haemoproteus and malaria infections in 3 species of birds. MAanwell (1955a) noted seasonal incidence of blood parasites in the song sparrow. Considerable year-to-year incidence data were reported by Herman et al (1954). The writer has accumulated data relative to

Hematozoa from Colorado Birds. I. Pigeons and Doves

A survey of the blood smears from 464 Colorado columbiforms included blood from: 86 domestic pigeons (Columba livia), 109 band-tailed pigeons (C. fasciata fasciata), and 269 western mourning doves (Zenaidura macroura marginella). Haemoproteus columbae was found in 57 of the band-tails and 164 of the mourning doves; H. sacharovi was in 76 of the band-tails and 163 of the mourning doves. Leucocytozoon marchouxi occurred in 1 of the domestic pigeons, 39 of the band-tailed pigeons, and 37 of the mourning doves. Plasmodium hexamerium was found in one of the domestic pigeons and in one of the mourning doves. Trypanosoma avium was seen in two of the mourning doves; microfilariae were found in one of the band-tails and in nine of the mourning doves. This is the first record of a natural infection with Plasmodium hexamerium in either the domestic pigeon or the western mourning dove, and the second reporting of Leucocytozoon marchouxi in the domestic pigeon. In 51 western mourning doves of which both the femoral bone marrow and blood were examined, Trypanosoma avium was found in the bone marrow of 13 and in the blood of none. Surveys of the blood parasites of birds are legion. Few surveys, however, have been made in which sizable numbers of birds of any one species have been examined. Furthermore, relatively few data are available from the Rocky Mountain region. During the past 15 years the senior author has collected approximately 2,000 blood films from Colorado birds. Rather than wait for all these films to be examined, it seemed best to report on each particular group of birds as its examination was completed. It was decided to start with the Columbiformes (pigeons and doves), with which this paper deals. A short abstract previously dealt with some of the band-tailed pigeons included here (Stabler, Limberg, and Matteson, 1950). MATERIALS AND METHODS Collected within some 30 miles of Colorado Springs, Colorado, the birds were either shot or captured alive. They included: 88 domestic pigeons (Columba livia), 109 band-tailed pigeons (C. fasciata fasciata), and 269 western mourning doves (Zenaidura macroura marginella). Blood films from the dead birds were made from the thoracic cavity; those from the live ones were made from a snipped toenail. Films were stained with Giemsa and searched carefully for hematozoa. The femoral marrow from 51 of the mourning doves was also examined in physiological salt solution for trypanosomes.

A Parasitological Survey of Fifty-One Eastern White-Winged Doves

In a very useful check-list of the parasites of pigeons and doves, Levine and Kantor (1959) cited four sets of authors who have listed parasites of the white-winged dove (Zenaida asiatica) : Laveran and Petit (1909), and Beltran (1942), who reported Haemoproteus only; Wood and Herman (1943), who found Haemoproteius (two species), Trypanosoma, and Plasmodium; and Saunders (1955), who noted the presence of microfilariae. The only other reference to the parasites of these doves is that of Saunders (1959), who reported on 72 of them in a survey of blood films from wild Mexican columbids. (See Addendum) Because of the additional data secured by the author in a parasitological study of 51 individuals of this dove, the present observations seem worth recording.

On the Presence of Peripheral Chromatin in Endolimax nana

Linmax-amoebae have been described from a large series of hosts, for example: oysters; cockroaches; termites; larvae of Phyllophaga sp., crane-flies and Harlequin flies; rock-fish; frogs; lizards; domestic fowl; domestic turkey; rats; man and others. In the descriptions of these various amoebae is found more or less general agreement regarding certain features of the nuclear structure. The karyosome is uniformly recorded as being of large size and staining intensely with hematoxylin. In some of the species spoke-radii are reported to connect the karyosome with the nuclear membrane. The greatest discrepancies, however, occur in the observations on the peripheral chromatin, some forms being recorded as having an abundance, while none was noted in others. In the form from man, Endolimiax nana, the nucleus is described as having a large, darkly staining karyosome, with spoke-radii extending to the nuclear membrane, on which is to be found no true peripheral chromatin. In view of the differences of opinion regarding the nuclear structure of the limnax-amoebae, some observations on the nucleus of Endolimax nana seemed worthy of presentation. The writer is grateful to Dr. D. H. Wenrich of the Department of Zoology of the University of Pennsylvania, for guidance and criticism. The material for these observations was obtained from numerous

Ingestion Processes on lodamoeba (Protozoa).

In discussing the food habits of Entamoeba muris and E. ranarum, Wenrich (1941) described the presence of differentiated pharynges and tubes which appeared to be used in ingestion. In 1943 he described similar structures on a flagellate, Histomonas meleagridis, from domestic chickens and pheasants (Phasianus torquatus). He recently (1944) noted them on Dientamoeba fragilis from man. The following observations were made on lodamoeba from man and two other primates. These structures were not noted by Wenrich (1937) in his paper on Iodamoeba biitschlii.

Parasitism of Mosquito Larvae by Mermithids (Nematoda).

Numerous species of fungi, bacteria, and worms have been recorded as parasitizing mosquitoes. Some have been shown to be definitely destructive of these insects (for a bibliography of the relevant literature up to 1927 see Speer, 1927). Among the worms, there are nematode parasites of adult mosquitoes, such as those described from Aedes sollicitans by Stiles (1903), by Smith (1904), and those from A. vexans females by Hearle (1926). These latter, collected first in 1920 and 1921 by Hearle, were called Paramermis canadensis by Steiner (1924), and were said to interfere with ovarian development. Stiles (1903), who was the first to describe a mermithid worm from a mosquito in North America, proposed the name Agamomermis culicis for his form. Other similar worms are described as parasitizing the larvae of land and fresh water arthropods, including mosquitoes, and are placed in the superfamily MERMITHOIDEA, family MERMITHIDAE (Chitwood, 1950). For a discussion of this group of parasites see Christie (1941), and Steinhaus (1949). Mermithid parasites of the larvae of mosquitoes were first noted from this hemisphere by the author (1945a and 1945b). They were observed in fourth instar larvae of Aedes vexans and Culex salinarius from Delaware Co., Penna., and were seen to be invariably fatal to their hosts. Jenkins, in a paper before the Amer. Assoc. Econ. Ent. at Denver in December, 1950, reported a similar worm from the larvae of arctic and subarctic mosquitoes (northern Canada and Alaska). Drs. Jenkins and West kindly allowed me to see their manuscript on these worms. They present a thorough review of the literature, and plan extensive field and laboratory studies.