Diamond Medium Research Articles

Drug Tolerance by Tritrichomonas foetus

A strain of Tritrichomonas foetus that remained refractory to dimetridazole, despite four courses of successively higher levels of medication (strain 4711, McLoughlin, 1965), was recovered from a bull and established intravaginally in hamsters. Tolerance for the chemical remained undiminished after maintenance of the strain in hamsters for 27 months. Also in hamsters, T. foetus that had no prior exposure to dimetridazole became unresponsive to the drug following pretreatment with suboptimal levels. The strain recovered from the bull and those from the hamsters had a detectable crossresistance to metronidazole and to aminitrozole. A previous report (McLoughlin, 1965) mentioned an apparent dimetridazole-resistant strain of Tritrichomonas foetus (now designated strain 4711) that had been recovered from Bull 4711. This animal had received a 5-day course of treatment at 25 mg/kg body weight admixed with the feed, and 5-day courses of treatment at 25, 50, and 100 mg/ kg, respectively, administered by capsule. The infection persisted and remained refractory to subsequent single intravenous injections of 20 and 30 mg/kg. The present report is a summary of data on the comparative response in hamsters to dimetridazole and other chemicals (Fig. 1) by this strain and a normal one that had no prior exposure to dimetridazole. The results of attempts to experimentally develop drug-resistant strains of T. foetus in laboratory animals are also reported. MATERIALS AND METHODS Tritrichomonas foetus recovered from Bull 4711 were cultured in Diamond's medium (Diamond, 1957) and then established intravaginally in hamsters essentially as suggested by Rubin and Cordray (1958). Periodically tubes of the same medium were inoculated with vaginal secretions from these hamsters, and the resulting cultures used to infect additional animals. On 7 occasions in 27 months from the time the strain was originally isolated, groups of usually 5 to 10 infected hamsters were given 100 mg/kg of dimetridazole1 per os for 3 or 5 days. Concomitantly, similar regimens were used in control groups of hamsters infected with a normal strain of T. foetus. Vaginal secretions from principal and control animals were examined microscopically and Received for publication 12 December 1966. 1Dimetridazole and metronidazole were supplied through the courtesy of Rhodia, Inc., New York, N.Y. by cultural techniques at least twice, usually 3 and 7 days after treatment. Other groups of hamsters infected with a normal strain of T. foetus were given suboptimal levels of dimetridazole for varying periods and then examined for trichomonads. Hamsters that were still positive for T. foetuis were given 50 or 100 mg/kg of dimetridazole per os for 3 or 5 days. The protocol for this portion of the study is given in Table II. On one occasion, 19 hamsters with intravaginal infections of normal T. foetus were given metronidazole orally for 5 days at a dose rate of 25 mg/kg body weight. Those that remained infected were given 100 mg/kg for 5 days and subsequently examined for trichomonads. In cross-resistance trials, groups of hamsters infected with strain 4711 or with an experimentally induced dimetridazole-resistant strain (EI) were given metronidazole (100 mg/kg) or aminitrozole (100 or 200 mg/kg) daily for 5 days. In one instance, an initial regimen of 100 mg/kg aminitrozole was followed by a second 5-day course of treatment at the 200 mg/kg level. In each trial, control groups of hamsters infected with a normal strain of T. foetus received medication identical with that of the corresponding principals. All ham-

Frozen storage of Tritrichomonas foetus for 5.6 years.

SYNOPSIS. Tritrichomonas foetus was cultivated in Diamond's medium without phosphates or agar; 10% glycerol was added to the medium and the protozoa were frozen slowly and stored at −28 or −95 C. About half of the trichomonads died within the first day at either temperature. Thereafter, they continued to die off slowly during storage at −28, and none remained alive at or after 1024 days. New cultures could not be initiated with them after 64 days. At −95, almost as many trichomonads were alive after 256 days as after 1 day. Thereafter, however, they decreased in numbers, but 11% were still alive after 2048 days (5.6 years). The survivors appeared in good condition, and new cultures were readily initiated with them.

Dimetridazole, a Systemic Treatment for Bovine Venereal Trichomoniasis. I. Oral Administration

Tritrichomonas foetus infections were eliminated from six bulls by the oral administration of dimetridazole at the rate of 50 mg/kg body weight daily for 5 consecutive days. The chemical was given either by capsule, or admixed with the feed. Five daily doses at 25 mg/kg administered by capsule freed another bull of T. foetus, but this level was ineffective when fed in grain to two bulls. One of the latter subsequently became negative after repeated courses of treatment at higher dosages. The other bull retained the infection. Bovine venereal trichomoniasis, according to Levine (1961), probably is the third-ranking cause of abortion in cattle. Secondary complications, such as endometritis and pyometra, also contribute to the economic losses occasioned by infections of Tritrichomonas foetus. Levine, in 1961, summarized the extensive literature on this parasite. Subsequent reports pertinent to the chemotherapy of the disease include those of Francis and Collins (1963), Fitzgerald et al. (1963), Gasparini et al. (1963), Rigoulet (1963), and McLoughlin (1963, 1964). The first two papers deal with topical application of therapeutic agents, and the others with the systemic use of imidazole derivatives. Although encompassing much of the material included in brief earlier notes (loc. cit.), this report presents more comprehensive and detailed observations on the oral use of dimetridazole (1,2-dimethyl-5-nitroimidazole) against T. foetus infections in bulls. MATERIALS AND METHODS Experimentally infected bulls were used throughout. The animals were anesthestized, or adequately tranquilized, the penis withdrawn from the sheath, scarified, and then massaged with the trichomonad culture. The animals were examined 1 week after exposure to the parasite and were reexamined prior to treatment to verify the infection. In no instance was an established infection spontaneously lost. Preand posttreatment examinations were made by pipetting physiological saline into the prepuce, closing the opening by hand, and massaging for several minutes. The washings were then collected, centrifuged, and portions of the sediment used to inoculate tubes of Diamond's medium (Diamond, 1957); other portions were examined microscopically for trichomonads. The cultures were examined after intervals of 48, 120, and 144 hr. Received for publication 5 April 1965. The animals were weighed at the last pretreatment examination. Dimetridazole (supplied through the courtesy of Rhodia, Inc., New York, N. Y.) was administered either by capsule with the aid of a balling gun, or admixed in the daily grain ration in dosages of 25 to 100 mg/kg body weight. Each course of treatment lasted 5 days. With the 50-mg dosage administered in the feed, it was necessary to mask the flavor of the medicated grain with molasses after the 2nd day, and finally to withhold all other feed after the 4th day to assure total consumption of the medicament. At least three negative posttreatment examinations during a period of 1 to 7 months were made before an animal was considered free of the disease. Two of the treated bulls were test-bred, and one animal was necropsied.

Axenic culture of Tetratrichomonas gallinarum: Growth and anaerobic utilization of carbohydrates and related compounds

Tetratrichomonas gallinarum was grown in Diamond's medium and in a medium containing gastric mucin, trypticase, yeast extract, dextrose or any utilizable sugar, ascorbic acid, cystine, and agar. The flagellate multiplied uniformly between 33 ° and 44 °C, the optimum temperature being 40 °. The growth rate was controlled by the temperature of incubation and the pH of the medium. The flagellate utilized dextrine, fructose, galactose, dextrose, inulin, lactose, maltose, raffinose, soluble starch, and sucrose. The flagellate is a different biochemical strain from that reported by Lindblom.

The Effect of Cultivation and Freezing on the Virulence of Trichomonas vaginalis for Mice

The effect of cultivation on the virulence of several T. vaginalis strains for mice injected intraperitoneally was studied. A comparison of 12 strains of T. vaginalis injected intraperitoneally into mice revealed loss of virulence in the majority of strains after 2 to 3 months cultivation that did not appear to be due to random variation. The sex of the mouse host was not a factor in virulence assay via the intraperitoneal route. Preliminary studies indicated that the virulence of a strain via the intraperitoneal route can be preserved by freezing the organism. The pathogenicity of Trichomonas vaginalis for laboratory animals has been investigated recently by Honigberg and Braunthal (1957), Iwai (1957), Reardon and Jacobs (1958), Bogovsky and Teras (1958), Newton et al. (1960), Reardon et al. (1961), Honigberg (1961), and Frost and Honigberg (1962). These workers have observed considerable differences in virulence of various T. vaginalis strains for laboratory animals. Previous studies have shown that many species of pathogenic organisms lose virulence upon cultivation. Whether this occurs with T. vaginalis has not been clearly established. Honigberg (1961) reported no loss of virulence by a T. vaginalis strain after 5 months of cultivation when injected subcutaneously into mice. The present study was undertaken to determine whether cultivation affected the virulence of T. vaginalis for mice injected intraperitoneally. Since these studies revealed that loss of virulence did occur, preliminary experiments were made on the effect of preservation by freezing on the virulence of T. vaginalis. MATERIALS AND METHODS Strains of T. vaginalis were isolated from vaginal smears placed in Diamond's culture medium (1957) containing 200 units of penicillin and 200 ,Ag of streptomycin per ml and were maintained at 35 C. Transfers to fresh medium were made every 48 hr. After six transfers the strains were inoculated into Received for publication 17 May 1963. * This investigation supported in part by U. S. Public Health Service Grant E-3593. 226 medium lacking antibiotics. After two subcultures in medium lacking antibiotics, the cultures were tested for bacterial contamination by inoculating tubes of brain-heart infusion and thioglycollate broth and observing for at least 1 week for the presence of bacteria. Only strains showing no evidence of bacteria contamination were used in these studies. All strains were tested initially for virulence between the 3rd and 4th week after isolation and again 2 to 3 months later. Twenty-fourto 30-hr-old cultures were used for all animal inoculations. The organisms were centrifuged at 375 g for 6 min and washed once in 5 ml of sterile 0.85% saline. After centrifuging again and removing the saline, the organisms were suspended in Diamond's medium minus serum. The suspension was then standardized by hemocytometer counts to 2 X 106 organisms per ml. White mice, 2 to 3 months old (National Lab, St. Louis, Mo.), were injected intraperitoneally with 106 organisms. The mice were observed for 2 weeks, deaths and gross pathological appearance of the dead being recorded during this interval. Checks for possible bacterial contamination of cultures used for inoculation were made using brain-heart infusion broth and thioglycollate broth. Also, the lesions of randomly selected experimental animals were checked for possible bacterial contamination. Strains of T. vaginalis that were to be frozen were inoculated into bottles containing 150 ml of Diamond's medium minus agar. After 48 to 72 hr incubation at 37 C in a nitrogen atmosphere the cultures were centrifuged at 375 g for 6 min and the supernatant fluid discarded. The organisms were suspended in fresh medium containing 10% glycerol to give a final concentration of 200 X 106 organisms per ml. The suspension was dispensed into small ampules in 0.1 to 0.2 ml amounts and the ampules sealed with a gas torch. The ampules were placed in a cotton-lined metal container and stored in a -43 C deepfreeze. The following day the ampules were transferred to precooled vacuum jars. After 8 weeks the frozen strains were thawed This content downloaded from 207.46.13.128 on Tue, 06 Sep 2016 04:36:42 UTC All use subject to http://about.jstor.org/terms LINDGREN AND IVEY-CULTURE AND VIRULENCE IN TRICHOMONAS 227 TABLE I. Effect of prolonged cultivation on the virulence of strains of Trichomonas vaginalis for mice injected intraperitoneally. Strains Weeks in Number of Average number days culture deaths for death to occur 1 3 0/10 1 12 0/10 2 3 6/10 4.7 2 12 3/10 8.3 3 3 7/10 8.6 3 15 1/10 13.0 4 3 3/10 8.7 4 15 6/10 10.0 7 3 5/10 9.6 7 15 5/10 9.4 8 3 9/10 10.3 8 15 1/10 13.0 9 3 8/10 8.5 9 15 1/10 13.0 11 3 0/10 11 15 2/10 12.0 and inoculated into fresh medium. Virulence assays were performed 1 week later.

The Morphology of Trichomonas ovis from the Cecum of Domestic Sheep

Trichomonas ovis (Robertson, 1932) Morgan, 1946 was found in the ceca of 12 out of 16 domestic sheep in Illinois and Utah and was cultivated readily at 37 C in Diamond's medium, CPLM medium and bovine cecal extract medium. It was studied by phase-contrast microscopy and after fixation with osmium tetroxide or Bouin's or Hollande's fixatives and staining with Giemsa, Heidenhain's iron hematoxylin, or silver impregnation. It was piriform, 6 to 9 by 4 to 8 A with a mean of 7 by 6 ,u, with an anterior nucleus, with a prominent pelta at the anterior end, with a slender, hyaline axostyle which protruded a mean of 5 , beyond the body and gradually tapered to a point, without a chromatic ring at the point of exit of the axostyle, with 4 anterior flagella of unequal length averaging 14, 11, 9, and 7 u, respectively, with a prominent undulating membrane which extended from three-fourths to the full length of the body and usually had two to three undulations, with a posterior, free flagellum extending beyond the undulating membrane, with a prominent costa and several irregular rows of paracostal granules, with an ovoid or club-shaped parabasal body averaging 2 by 1 a and containing an intensely chromophilic body, and with a parabasal filament. This is the only species of enteric trichomonad of sheep or cattle which has been described in detail following study by modern techniques. Robertson (1932) described a trichomonad which he observed in the gut in one of 86 sheep in England. He said that this trichomonad possessed only two anterior flagella and therefore proposed the name Ditrichomonas ovis for it. Since that time there appears to have been no further investigation of this parasite or of any other trichomonad from sheep. In 1958, Dr. Krishna N. Mehra of the College of Veterinary Medicine, University of Illinois, Urbana, Illinois, isolated a strain of cecal trichomonad from a domestic sheep (Ovis aries). This strain, labeled 0, has been used since that time in connection with experiments on the survival of various trichomonad species during extended frozen storage. This strain possessed four anterior flagella and also differed in other morphological aspects, such as body size and prominence of the undulating memReceived for publication 15 March 1962. * This investigation was supported in part by a research grant (E790C) from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, and in part from a Utah State University Research Fellowship, Utah State University, Logan, Utah. t Present address: Department of Zoology, Utah State University, Logan, Utah. brane, from the trichomonad described previously by Robertson. Four other strains which were subsequently isolated from sheep during 1961 (one from a sheep raised in Illinois, one from a sheep shipped to Illinois from Texas, and two from sheep killed in Logan, Utah) all appeared identical with strain 0 when observed with the phase-contrast microscope. Because of the obvious differences from the trichomonad described by Robertson as Ditrichomonas ovis, it was decided to make a detailed study of the morphology of these cecal trichomonads. MATERIALS AND METHODS All observations reported in this paper were made on trichomonads of strain O. Cultures were grown at 37 C in Diamond's (1957) medium without agar or phosphate buffer, in Johnson and Trussell's (1943) CPLM medium, and in bovine cecal extract medium (Jensen, 1961), which was a modification of the cecal extract medium used by Hibler et al. (1960) in their investigation of porcine trichomonads. A Zeiss photomicroscope was used for both phase-contrast and bright-field observations. Morphological studies were based upon observations of living trichomonads, of specimens fixed for one minute in the vapors of 2% osmium tetroxide solution, and of specimens stained with Giemsa's stain

Survival of Tritrichomonas foetus stored at -28 and -95 degrees C after freezing in the presence of glycerol.

SYNOPSIS. Tritrichomonas foetus survived much better on extended storage at ‐95 than at ‐28d̀C following slow freezing in the presence of 1.0 M glycerol. There was no significant difference between these temperatures in survival up to 8 days, but thereafter the protozoa continued to die off slowly at ‐28d̀, whereas their numbers remained essentially constant at ‐95d̀ for 128 to 256 days. The trichomonads' motility was much better after storage at ‐95d̀ than after storage at ‐28d̀, and fresh cultures could be initiated from the former much more readily.Other constituents of the suspending medium besides glycerol affect the survival of the protozoa upon freezing. Survival was much better when the protozoa were frozen in the original Diamond's trypticase‐yeast extract‐maltose‐cysteine‐ascorbic acid‐serum medium in which they had been grown than when they were frozen in physiological salt solution or in fresh Diamond's medium. There was no significant difference between survival in the latter two suspending media. The speed and time of centrifugation needed to remove the trichomonads from the medium in which they had been grown had no effect on their survival upon subsequent freezing. Presumably some product or products of the trichomonads' metabolism have an additional protective action which supplements that of glycerol.When frozen in the original Diamond's medium in which they had been grown plus 1.0 M glycerol, an average of 15% of the trichomonads were alive after 128 days' storage at ‐28d̀ and an average of 38% were alive at ‐95d̀C. When frozen in physiological salt solution plus 1.0 M glycerol, these percentages were 8% and 12% respectively.

Hypotrichomonas acosta (Moskowitz) from Reptiles. 11. Physiology.*

SYNOPSIS.Three strains of Hypotrichomonas acosta were isolated in axenic culture. Attempts to develop a defined medium directly from a defined medium suitable for Tetrahymena pyriformis were unsuccessful. Development of partly defined media by substitution for undefined materials in Diamond's medium were more successful. Horse serum was replaced by 1 mg. % TEM‐4T (a diacetyl tartaric acid ester of monoglycerides from tallow) and 0.5 mg.% cholesterol. Yeast extract was replaced by a mixture of ribonucleotides. Inclusion of several additional components permitted reducing the Trypticase concentration from 2% to 0.25%.