Barn Strain Research Articles

ATTEMPTS AT INFECTING RINGED TURTLE DOVES WITH VIRULENT Trichomonas gallinae

Twenty Trichomonas -free ringed turtle doves ( Streptopelia risoria ) were inoculated per os with the highly virulent Jones' Barn strain of Trichomonas gallinae . None became infected. Three F1 females housed together were similarly inoculated with this strain and remained Trichomonas -positive for upwards of 182 days. They showed no disease and eventually lost their infections. These three positive females "mated" and laid several six-egg sets in a communal nest. At successive nestings they were given: 1) a fertile domestic pigeon ( Columba livia ) egg, and 2) two fertile ringed dove eggs, all of which hatched. The pigeon squab died of trichomoniasis on day four; the doves survived to maturity. When trichomonads from these doves were placed in Trichomonas -free domestic pigeons the latter all died of T. gallinae trichomoniasis on postinoculation day 8.1 (average).

Effect of Different Immunization Procedures on Agglutination and Precipitation Reactions of Trichomonas gallinae

Immune sera were prepared in rabbits by intravenous or subcutaneous inoculations of an avirulent T. gallinae substrain JB(VI)C, derived from the highly pathogenic Jones' Barn strain by prolonged in vitro cultivation. Living or disrupted antigen was used for intravenous injections, while only disrupted organisms were administered via the subcutaneous route. In some rabbits inoculations of the antigen were given in combination with the complete Freund's adjuvant. The highest agglutination and hemagglutination titers, but the fewest and weakest precipitin lines on gel diffusion plates, were observed with sera from rabbits inoculated intravenously with living flagellates. In contrast, the lowest agglutination and hemagglutination titers and the most numerous and strongest precipitin lines were recorded with sera from rabbits which received disrupted trichomonads via the subcutaneous route. Sera prepared by several other variations of the immunization method fell between the above two in their agglutinating, hemagglutinating, and precipitating capacities. The employment of the complete Freund's adjuvant appeared to enhance somewhat the strength of all immune sera, its effects being most clearly evident in the results of gel diffusion experiments. A survey of the literature reveals that various forms of agglutination have been the methods used most extensively in immunologic studies of trichomonads (Honigberg, 1970). In many investigations the entire immunization series involved intravenous inoculations of living parasites into rabbits; in at least one study (Samuels and Chun-Hoon, 1964) only part of the immunizing series was administered by this method. The superiority of living flagellates injected intravenously into rabbits in stimulating production of agglutinating antibodies to Trichomonas vaginalis was suggested by the comparative studies of Trussell (1946) and Teras (1961, 1962). Much lower titers were obtained with sera of animals which received intravenous injections of killed organisms, and the results of subcutaneous or intramuscular inoculations were still less satisfactory. No extensive comparative investigations have been reported with respect to any other trichomonad species, but according to Robertson (1941, 1960), living Received for publication 22 June 1970. * This investigation was supported by Research Grants AI00742-13, 14, from NIAID, U. S. Public Health Service. t Dr. Stepkowski, Associate Professor of Veterinary Science and Chief of the Laboratory of Poultry Diseases, College of Agriculture, Lublin, Poland, was a Guest Scientist in the Department of Zoology, University of Massachusetts at Amherst, from January 1968 to March 1969. Tritrichomonas foetus were more effective than dried flagellates in the production of agglutinating antibodies. Nonetheless, satisfactory agglutination, hemagglutination, precipitation, and/or fluorescent antibody reactions with trichomonads were obtained by several workers who used killed trichomonads, prepared in various ways, for immunization of rabbits and other animals (Morisita, 1939; Kerr and Robertson, 1943, 1954; MacDonald and Tatum, 1948; Osaki and Oka, 1962; Hoffmann and Gorczynski, 1964; Stepkowski, 1966; Goldman and Honigberg, 1968; Honigberg and Goldman, 1968). Subcutaneous inoculations of disrupted Trichomonas gallinae in the presence of adjuvant gave very strong rabbit sera for precipitation and fluorescent antibody tests (Goldman and Honigberg, 1968; Honigberg and Goldman, 1968), and the advantages of using adjuvant in immunization were brought out also by agglutination experiments with Tritrichomonas augusta (Samuels and Chun-Hoon, 1964). The present investigation was undertaken to ascertain which immunization method would give the most satisfactory rabbit sera for agglutination, hemagglutination, and precipitation (gel diffusion) reactions involving T. gallinae. MATERIALS AND METHODS

Passive immunization of pigeons against trichomoniasis.

SYNOPSIS Nonimmune homing pigeons Columba livia were infected with the Jones' Barn strain of Trichomonas gallinae and subsequently transfused with plasma from acute or chronically infected pigeons harboring one of 3 different strains of T. gallinae. The transfusions were either a single 2 ml dose given one day after inoculation or three 1 ml doses given 0, 5, and 10 days after inoculation. Plasma from pigeons harboring any of the 3 strains was capable of passively immunizing nonimmune birds. All birds which were immunized with plasma from infected pigeons survived until killed at the end of the test period and no visceral lesions were found on necropsy but trichomonads were present in the oropharynx. All controls (untreated or transfused with normal plasma) died of visceral trichomoniasis.Immune plasma produced some lysis of trichomonads in vitro, and inhibition of motility and vacuolization occurred in some of the non‐lysed organisms. The overall lytic activity in vitro affected less than 10% of the suspended trichomonads.

Further Observations on the Effects of Various Laboratory Procedures on the Virulence of Trichomonas gallinae for Pigeons

With dimethyl sulfoxide as the cryoprotectant, axenic cultures of three isolates (JBIV, JBV, JBVI) of the Jones' Barn (JB) Trichomonas gallinae strain retained their original virulence for pigeons after 5, 5.5, and 7 years of storage at subzero temperatures, primarily in liquid nitrogen at -196 C. The closely comparable times required by noncloned and cloned populations of the JBVI trichomonads to kill Trichomonas-free, nonimmune pigeons indicated the homogeneity of the JB strain with respect to virulence. Although higher pathogenicity for birds was retained by the JBVI parasites maintained for 1 year by serial intraperitoneal passages in mice than by those kept for the same length of time in nonliving media, it was much lower than the virulence typical of the JB strain. The flagellates transferred in mice did not kill the avian hosts and their original pathogenicity levels could not be restored by several bird-to-bird passages. Nearly full virulence for pigeons was retained by the JBVI isolate which had been maintained for up to 1 year in the presence of chick liver cell cultures. Cell culture medium-CPLM mixture had no virulence-preserving effect. JBV trichomonads kept for 22.5 weeks in a CPLM-type medium, which contained no added carbohydrates and in which little division of the parasites took place, appeared to retain their pathogenicity for birds for a longer time than when maintained in the conventional CPLM and fluid thioglycollate media. The first report on the effects of various laboratory procedures on the virulence for pigeons of the Jones' Barn (JB) strain of Trichomonas gallinae (Rivolta) summarized the experimental results obtained during the early stages of our study (Stabler et al., 1964). According to these results: (1) prolonged (19 to 21 weeks) axenic cultivation of the JB parasites by serial transfers in nonliving media at 37 + 0.5 C caused pathogenicity loss; (2) trichReceived for publication 12 March 1970. * This investigation was supported by Research Grant AI00742, from the NIAID, U. S. Public Health Service. t Department of Biology, Colorado College, Colorado Springs, Colorado 80903. t Permanent address: Department of Protozoology, Institute of Parasitology, Czechoslovak Academy of Sciences, Prague, Czechoslovakia. Dr. Kulda spent 2 years as Research Associate in the Department of Zoology, University of Massachusetts at Amherst. During this period he was supported mainly by Research Grants AI00742-13, 14, from NIAID; part of his support was provided by Biomedical Sciences Support Grant 5-SO5-FR07083-03, U. S. Public Health Service. ? The help of Mrs. Mary Tarr Hadley in certain parts of this investigation is hereby acknowledged. omonads stored at -20 or -72 C for 52 weeks retained their original virulence level; (3) pathogenicity loss was enhanced by the inclusion of penicillin and streptomycin in the original isolation medium (the JB flagellates ceased to kill nonimmune pigeons after 9 weeks of cultivation at 37 ? 0.3 C). These findings, which served as the basis for subsequent immunologic (Goldman and Honigberg, 1968; Honigberg and Goldman, 1968; Stepkowski and Honigberg, in Honigberg, 1969) and biochemical (Honigberg and Livingston, 1968) studies, suggested undertaking additional investigations of the ways whereby different laboratory procedures might affect the virulence of the JB strain. The present paper deals with the results obtained in the course of these investiga-

Effect of Certain Laboratory Procedures on the Virulence of Histomonas meleagridis for Turkeys and Chickens

The Hm-L1 strain of Histomonas meleagridis was found to be highly virulent for young chickens and turkeys, which died 9 to 10 days after infection, on the average. In vitro cultivation of the parasites at 41.5 C for 9 weeks was accompanied by a gradual attenuation of pathogenicity, and no birds were killed by the end of that period. The original virulence levels could be restored for up to 15 weeks of cultivation by chicken-to-chicken or turkey-to-turkey passages; after that time they could be restored no longer. As evidenced by the increase in the length of time between inoculations and death of the chickens, pathogenicity was slightly lower in flagellates isolated from carrier hosts. Original virulence levels were restored, however, to these parasites after 4 serial passages in chickens. No decrease in virulence occurred in the Hm-L1 histomonads maintained in liquid nitrogen (-196 C) for 20 weeks from the time of their isolation. Preliminary experiments with the Hm-LI strain of Histomonas meleagridis suggested that it was about equally virulent for turkey poults and for young chickens, causing a disease accompanied by very similar lesions in both host species and killing the birds within a relatively short time. Various aspects of pathogenicity of the Hm-LI histomonads, including its in vitro attenuation, appeared to resemble those of the Jones' Barn (JB) strain of Trichomonas gallinae (Stabler et al., 1964). In view of the unusually high virulence of the Hm-LI parasites for chickens, which are more readiy available and easier to maintain than turkeys, and because of the significant amount of information on the methods of pathogenicity preservation and restoration with respect to the JB strain of T. gallinae (Stabler et al., 1964; Honigberg et al., MS), it was decided to employ the Hm-Li strain for a study of various aspects of pathogenicity in H. meleagridis by such methods. The present report deals with the results of this study. MATERIALS AND METHODS Strains of histomonads A strain of H. meleagridis, isolated from turkeys in March 1968 by Dr. Everett E. Lund (Beltsville Parasitological Laboratory, Animal Disease and Received for publication 30 December 1969. * This investigation was supported by Research Grants AI00742-14, 15 and Training Grant TOI-AI-00226-07, from the NIAID, U. S. Public Health Service. t A short report of this study was given at the 22nd Annual Meeting of the Society of Protozoologists (Dwyer and Honigberg, 1969). Parasite Research Division, Agricultural Research Service, U. S. Department of Agriculture, Beltsville, Maryland), was sent by him to Amherst 1 year later. According to Dr. Lund (pers. comm.), the strain, designated by us as Hm-LI, had high infectivity ( 95%) for, and caused a fatal disease in ~90% of the infected turkeys. The original pathogenicity level of the strain was maintained by serial passages through these birds. Parasites derived from the passages were used for inoculating turkeys and chickens which served as controls for all experiments. In vitro cultivation Cecal wall scrapings and lumen contents of turkeys which died from infection with the Hm-L1 strain were suspended in sterile 0.85% w/v NaCl solution and incubated for 0.5 hr at 41.5 ? 0.5 C. During incubation the mixture was agitated at 10-min intervals to free the parasites from the debris. Subsequently, 1-ml aliquots of the suspension were inoculated into 20 ml (115 by 20 mm) flat-bottom, screw-cap test tubes, each containing 9 ml of Dwyer's (1970) culture medium. The agnotobiotic (xenic) cultures, incubated at 41.5 0.5 C, were transferred according to the schedule recommended by Dwyer (1970).

Serum Protein Changes in Immune and Nonimmune Pigeons Infected with Various Strains of Trichomonas Gallinae

Serum protein changes were studied in immune and nonimmune pigeons infected with three different strains of Trichomonas gallinae. Strain I (nonvirulent) produced no change in the relative concentration of serum components. Strains II (oral canker) and III (Jones' Barn) produced decreases in albumin and alpha globulins, and increases in beta and gamma globulins between the 7th and 20th days post infection. Birds infected with strain II began to return to normal by the 20th day, while all those infected with strain III were dead between 10 and 14 days post infection. Two serum protein patterns resulted from infection of immune birds with the Jones' Barn strain. One showed no change in relative protein concentrations and no tissue invasion by the parasite while the other was similar to that seen in nonimmune birds infected with a strain producing oral canker. These also showed evidence of tissue invasion by the parasite. It was concluded that tissue invasion was necessary to evoke a quantitative change in serum protein concentrations.

Challenge Infection as a Means of Determining the Rate of Disease Resistant Trichomonas Gallinae-Free Birds in a Population

Trichomonas gallinae-free pigeons and mourning doves were infected with the Jones' Barn strain of T. gallinae to determine the rate of disease resistant T. gallinae-free birds in each population. Although all birds became infected 88% of the pigeons were resistant to trichomoniasis while 82% of the mourning doves were resistant. It was concluded that these birds had been previously infected and spontaneously lost their trichomonad fauna while retaining their resistance to fatal infection.

A Method for Producing Healthy Carriers of the Jones' Barn Strain of Trichomonas gallinae

A Method for Producing Healthy Carriers of the Jones' Barn Strain of Trichomonas gallinae

Immunologic analysis by gel diffusion technics of the effects of prolonged cultivation on Trichomonas gallinae.

SYNOPSIS. Antisera were developed in rabbits against 3 axenic lines of Trichomonas gallinae: JB(VI), the 6th isolate of the very virulent Jones' Barn strain, which was kept frozen in liquid nitrogen and had its full pathogenicity for pigeons when it was employed for immunization; JB(VI)C, a substrain derived from JB(VI), but attenuated during continuous in vitro cultivation for 1 year; and JB(V)C, a substrain of the 5th isolate of the Jones' Barn strain attenuated during continuous in vitro cultivation for over 3 years. All antisera were reacted on gel diffusion slides with varying concentrations of homologous and heterologous antigens. Two groups of precipitin bands, arbitrarily labeled A and B, were seen on the slides. Analysis of these bands revealed the common genetic makeup of the 3 trichomonad lines with respect to the group A bands. However, the group B antigenic system was strong in the attenuated JB(VI)C and JB(V)C substrains, and very weak in the fully pathogenic JB(VI) strain. These differences are discussed in the light of their possible relationship to pathogenicity.

Immunologic analysis by quantitative fluorescent antibody methods of the effects of prolonged cultivation on Trichomonas gallinae.

SYNOPSIS. Antisera were developed in rabbits against homogenized antigens prepared from 3 axenic lines of Trichomonas gallinae: JB(VI), the 6th isolate of the very virulent Jones' Barn strain which was kept frozen in liquid nitrogen and was fully pathogenic for pigeons at the time at which it was employed for immunization; JB (VI)C, a substrain derived from JB(VI), but attenuated during continuous in vitro cultivation for 1 year; and JB(V)C, a substrain of the 5th isolate of the Jones' Barn strain attenuated during continuous in vitro cultivation for over 3 years. Globulin fractions of the antisera were conjugated with fluorescein isothiocyanate (FITC), and fractionated on DEAE‐cellulose columns to yield highly specific fractions. Intact, formalin‐fixed flagellates of each line were stained on slides with each labelled antiserum under standardized conditions. Fluorescence emitted by individual organisms was measured with the aid of a special microfluorimetric apparatus. Experiments involving direct staining, one‐step inhibition, and cross‐absorption methods indicated the presence of unique and cross‐reacting antigenic systems in each of the 3 lines of trichomonads. The 2 substrains attenuated in the course of prolonged in vitro cultivation, altho antigenically similar, could be readily differentiated from each other. Further, both of them appeared to contain more antigenic systems than the still fully virulent JB(VI) parasites.

Cytochemical Observations on Chick Liver Cell Cultures Infected with Trichomonas vaginalis. I. Nucleic Acids, Polysaccharides, Lipids, and Proteins

Nucleic acids, polysaccharides, lipids, and proteins were studied in trypsin-dispersed chick liver cell cultures infected with a relatively pathogenic strain of Trichomonas vaginalis. The results were as follows. As the infection progresses fibroblasts and epithelial cells gradually lose their cytoplasmic RNA. The RNA content of the parasite-free experimental macrophages remains relatively constant, but it is significantly lower in the phagocytes with trichomonads closely applied to their external surfaces and in those which contain the flagellates within their cytoplasm. Nuclear DNA level of all cell types appears to show no significant changes throughout the experiments. The fibroblasts contain no stored glycogen, but there is much of this polysaccharide in the cytoplasm of the epithelial cells and moderate amounts are found in the macrophages. With time there is progressive depletion of glycogen stored in the epithelial cells in the experimental cultures, but the level of the carbohydrate remains fairly constant in the parasite-free macrophages. Significant loss of glycogen is seen in phagocytes invested with trichomonads and in those with engulfed flagellates. No acid mucopolysaccharides were demonstrable by the method employed in this study. Progression of infection is accompanied by a large accumulation of lipids in the fibroblasts and epithelial cells and by somewhat smaller fatty changes in the parasite-free macrophages. Significant loss of lipids is seen in phagocytes with trichomonads closely applied to their external surfaces and in those containing the protozoa within their cytoplasm. Most of the lipids present in all culture cells are neutral fats, although small amounts of unsaturated lipids can also be demonstrated. No phospholipids could be shown by the methods employed in this study. A weak reaction for proteins (Millon's test), localized primarily in small cytoplasmic granules, was noted in all cell types. The intensity of the reaction appeared to increase in the epithelial cells and macrophages in experimental cultures. The latter two cell types contain certain PAS-positive, amylase-resistant, sudanophilic granules which may be the same as those that give a positive reaction with Millon's test. These granules, judged to be probably glycolipoprotein in nature, seem more numerous in the experimental than in the control macrophages. The studies of behavior and pathogenicity of trichomonad flagellates in cell cultures (for a complete list of references see Honigberg et al., 1964, and Farris, MS.) have provided much important cytologic information upon the interaction of the protozoan parasites and the vertebrate cells. It has been established through these investigations that the pathogenic strains of Trichomonas vaginalis, a widely distributed species parasitic in the urogenital tract of man, and of Trichomonas gallinae, found in various birds, but most commonly in pigeons, cause profound pathologic changes in mammalian and avian cells maintained in vitro. Further, some insight has been gained into the mechanisms whereby the flagellates may cause injury to their hosts. Received for publication 17 December 1965. * This investigation was supported by Research Grant AI-00742 from the NIAID, U. S. Public Health Service. t Address during the academic year 1965-66: Laboratory of Parasitic Diseases, NIAID, Bethesda, Maryland 20014. To understand further the nature of the cell response to the presence and activity of T. gallinae, Abraham and Honigberg (1965) conducted a cytochemical study of the changes in content of nucleic acids, glycogen, and total lipids in trypsin-dispersed chick liver cell cultures exposed to the very pathogenic Jones' Barn strain of the avian parasite. The present account deals with the first phase of a similar, but more extensive, investigation undertaken to elucidate the cytochemical changes in cell cultures infected with a pathogenic strain of T. vaginalis. This paper reports the findings on nucleic acids, glycogen, other PAS-positive materials, mucopolysaccharides, lipids, and tyrosine-containing proteins. In subsequent publications we intend to deal with the various

Cytochemistry of Chick Liver Cell Cultures Infected with Trichomonas gallinae

A study of trypsin-dispersed chick liver cell cultures infected with the virulent Jones' Barn strain of Trichomonas gallinae was made by several cytochemical methods. A decline in RNA and RNP is apparent from the 8th hr following inoculation of parasites and becomes pronounced with time. The DNA seems unchanged during the entire experiment. No changes in RNA or DNA were observed in the controls. Significant decrease in glycogen in the epithelial cells becomes evident at 8 hr postinoculation, and after 16 hr this polysaccharide can no longer be detected cytochemically. The controls show no change in glycogen levels. The foregoing changes in the infected cultures are paralleled by an accumulation of lipids in all the cell types. The macrophages and to a lesser extent the epithelial cells contain certain PAS-positive sudanophilic granules, very likely glycolipoprotein in nature, which are probably associated with phagocytosis. The high pathogenicity of the Jones' Barn (JB) strain of Trichomonas gallinae for birds, especially pigeons (Stabler, 1954), mice (Honigberg, 1961; Frost and Honigberg, 1962), and cell cultures (Honigberg et al., 1964) has been well established. In trypsin-dispersed chick liver cell cultures, the parasites cause degeneration of the epithelial and fibroblast-like cells and stimulate activity of the macrophages. The flagellates are phagocytized by the macrophages, within which they multiply. Further, they appear to invade some of the epithelial and fibroblast-like cells (Honigberg et al., 1964). In view of these findings it was thought worthwhile to evaluate the effects of the JB strain on the cell cultures with the aid of cytochemical methods. The present study, which constitutes the first phase of the proposed investigation, deals with observations made on the nucleic acids, glycogen, other PAS-positive material, and lipids in normal and infected trypsin-dispersed chick liver cell cultures. A preliminary report of the study was given at the AIBS meetings at Boulder, Colorado in August 1964 (Abraham and Honigberg, 1964). MATERIALS AND METHODS The Jones' Barn strain, isolated originally by Dr. R. M. Stabler, was employed in this study. The Received for publication 15 February 1965. * This investigation was supported by Research Grant AI-00742 from NIAID, U. S. Public Health Service, and a Teachers Research Grant from the University of Massachusetts. tPermanent address: Osmania University, Hyderabad, India. details of the history of this strain are found in various reports of Stabler (see Stabler, 1954). In all experiments the December 1962 isolate was used (see Honigberg, 1961). The trypsin-dispersed liver cell cultures were prepared and the experiments set up according to the method described by Honigberg et al. (1964). Earle's BSS was used in preparation of the media and for washing cultures. In all instances 5 X 105 organisms from 24-hr trichomonad cultures were inoculated in 0.33 ml of CPLM medium without agar into the experimental cultures, while similar aliquots of fresh CPLM were added to the control cultures. The total volume of the medium in all cultures was 1 ml and the medium was adjusted to about pH 7.5 with the aid of 5% CO2 in air. The experimental and control preparations (on 9 to 10 by 22 mm cover glasses), at least two of each, were removed and fixed at intervals of 1, 2, 4, 6, 8, 10, 12, 16, and 20 hr. At each time interval the pH of the medium was also checked. The cell cultures were washed in warm Earle's BSS for at least 5 min before processing them for cytochemical studies. This proved helpful in removing debris accumulated from the medium which otherwise might have given false reactions with the dyes. For the study of the deoxyribonucleic and ribonucleic acids (DNA and RNA), some of the cells were fixed in a cold mixture of diethyl ether and absolute alcohol (1 : 1), stained according to the acridine-orange technique of Bertalanffy and Bertalanffy (1961), and examined under a fluorescence microscope (exciter filters Corning no. 5970 or 5840). May-Griinwald-Giemsa stain was employed for demonstration of deoxyriboand ribonucleoproteins (DNP and RNP), while Azure A-Schiff (Himes and Moriber, 1956) was used for DNA after fixation in Carnoy's fluid. Ribonuclease-treated preparations were employed as controls in the study of RNA. Glycogen and certain carbohydrate-containing materials were demonstrated by the periodic acidSchiff technique (PAS) of McManus (see Pearse, 1961) and counterstained with celestine blue and Meyer's hemalum, after fixation in cold acetic

THE BEHAVIOR AND PATHOGENICITY OF TWO STRAINS OF TRICHOMONAS GALLINAE IN CELL CULTURES.

SYNOPSIS. A comparison of the effects upon trypsin‐dispersed chick liver cell cultures of a virulent (Jones' Barn) and a non‐pathogenic (Lahore) strain of Trichomonas gallinae revealed significant differences in behavior of the parasites in cell cultures and in the response of such cultures. The virulent strain multiplies faster in nutrient medium in the presence of cell cultures; stimulates great activity of the macrophages; is not handled effectively by these phagocytes in which it can multiply causing their ultimate destruction; is found significantly more often within the cytoplasm of the liver epithelial and fibroblast‐like cells; causes very much more profound degenerative changes in all the cells, both invaded and non‐invaded; and suppresses effectively the division rate of the fibroblast‐like cells. On the other hand, the nonpathogenic strain multiplies at a lower rate in the presence of cell cultures; stimulates less activity of the macrophages; is handled readily by these phagocytes in which it multiplies only very rarely, if ever; is found seldom within the liver epithelial and fibroblast‐like cells; causes far less degeneration of all the cell culture elements; and suppresses significantly less the dlvision rate of the fibroblast‐like cells.At the end of a 20–24 hour period typically only a few living cells are left in cultures exposed even to attenuated isolates of the virulent strain, whereas those inoculated with the mild one do not show much degeneration even after 28 hours. The effects upon the cell cultures of cell‐free filtrates of actively growing trichomonad cultures are relatively minor, but the changes caused by the filtrates of cultures of Jones' Barn strain appear to be more extensive than those caused by similar filtrates of Lahore strain.

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-

Histopathological Changes in the Domestic Pigeon Infected with Trichomonas gallinae (Jones' Barn Strain)

Histopathological Changes in the Domestic Pigeon Infected with Trichomonas gallinae (Jones' Barn Strain)

Success of Soluble 2-Amino-5-Nitrothiazole in the Treatment of Trichomoniasis in the Domestic Pigeon

ABSTRACT INTRODUCTION THE person who manages pigeons in flocks sometimes needs a cure for canker (trichomoniasis due to T. gallinae) which can be administered through the food or drink. The therapeutic value of 2-amino-5-nitrothiazole (Enheptin—referred to here as ANT) was demonstrated by Stabler and Mellentin (1951 Stabler and Mellentin (1953). They gave it in gelatin capsules, pushed down the recipients’ throats. The soluble form of ANT (see below) was used with success by Fritzsche (1955) in Europe. He recommended a 0.125% solution for 6 days. The following experiments were carried out in order to secure further information on the therapy of canker via the drinking water. MATERIALS AND METHODS The trichomonads were the highly virulent Jones’ Barn strain of T. gallinae. The pigeons were homers (350 gm. av.) from the senior author’s Trichomonas-free loft. The drug was commercial Enheptin (ANT) soluble (45% 2-amino-5-nitrothiazole), manufactured by Lederle Laboratories Division, American Cyanamid Company. The birds . . .