- Email: [email protected]
Chromosome numbers of Tritrichomonas foetus and Tritrichomonas suis
Veterinary Parasitology 78 (1998) 247±251
Chromosome numbers of Tritrichomonas foetus and Tritrichomonas suis Wei-Dong Xu, Zhao-Rong Lun, Alvin Gajadhar* Centre for Animal Parasitology, Canadian Food Inspection Agency, 116 Veterinary Road, Saskatoon, Saskatchewan S7N 2R3, Canada Received 30 January 1998; accepted 27 March 1998
Abstract Tritrichomonas foetus and Tritrichomonas suis isolates were cultivated axenically in Diamond's medium. Studies on the chromosome numbers of these two species with a light microscope were done by adding different concentrations of colchicine into the medium, incubating at 378C for 6±8 h and using a hypotonic swelling technique. The diploid chromosome numbers of both T. foetus and T. suis were 2n10. # 1998 Elsevier Science B.V. All rights reserved. Keywords: Chromosome; Tritrichomonas foetus; Tritrichomonas suis
1. Introduction Bovine trichomoniosis is a venereal disease of cattle associated with infertility and abortion. The etiological agent is Tritrichomonas foetus, a flagellated protozoan parasite which is the cause of significant economic losses in the free-ranging cattle industry around the world (BonDurant et al., 1990; Speer and White, 1991). Tritrichomonas suis is a closely related morphologically indistinguishable flagellated protozoan found in the nasal passages, stomach, cecum and colon of swine (Hammond and Fitzgerald, 1953; Fitzgerald et al., 1958). Tritrichomonas suis (Gruby and Delafond, 1843), and T. foetus (RiedmuÈller, 1928; Wenrich and Emmerson, 1933) were originally isolated from pigs and cattle, respectively, and given separate species names. However, the taxonomical relationship between T. suis and T. foetus needs to be resolved because T. suis isolated from pigs established infections in both heifers and bulls (Fitzgerald et al., 1955; * Corresponding author. Tel.: +1 306 9755344; fax: +1 306 9755711; e-mail: [email protected] 0304-4017/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved PII S 0 3 0 4 - 4 0 1 7 ( 9 8 ) 0 0 1 5 0 - 2
248
W.-D. Xu et al. / Veterinary Parasitology 78 (1998) 247±251
Hammond and Leidl, 1957), and characteristics of in vitro cultivation, morphology and DNA sequences between T. foetus and T. suis showed remarkable similarities (Hibler et al., 1960; Jensen and Hammond, 1964; Felleisen, 1997). Theoretically, the chromosome number in the normal genome within any species of protozoa should be constant and variation may indicate species difference. Yuh et al. (1997) reported that six chromosomes were found in Trichomonas vaginalis, the human trichomonad. However, there are no reports on the chromosome numbers of T. foetus or T. suis. The purpose of the present study was to determine the numbers of chromosomes of T. foetus and T. suis and provide an indication of their genetic relationship. 2. Materials and methods Five isolates of T. foetus and three of T. suis were used in the present study (Table 1). Both T. foetus and T. suis were axenically cultivated in Diamond's medium (Diamond, 1957) modified by omitting agar and complementing with 50 mg mlÿ1 gentamicin, 1.0 mg mlÿ1 streptomycin and 10% inactivated newborn lamb serum at 378C. Colchicine and potassium chloride (KCl) treatments were similar to the methods described by Monk (1987), and Yuh et al. (1997) with slight modifications. Prewarmed (378C) solutions of 100 mM colchicine (Sigma) were added to the media cultivating T. foetus or T. suis when the concentration of the parasites reached 1.0±1.5106 organisms per ml. A series of concentrations of colchicine (0.125, 0.25, 0.5, 1.0, 2.0, 4.0 mM) in the media were used to determine a suitable concentration for each isolate to suppress cell division by inhibiting mitosis. Trichomonads were harvested from the media at 4±8, 10, 12, 14, 16, 20, and 24 h after colchicine was added. The culture was centrifuged at 200g for 10 min at room temperature, the pellets were washed once with phosphate-buffered saline (PBS, pH 7.3, 378C), and 10 ml of prewarmed (378C) 75 mM KCl was slowly added to each tube, and the pellets gently resuspended with a pipette and incubated at 378C for 12± 90 min (Table 1). The optimal duration of KCl treatment was determined by microscopic Table 1 Isolates of Tritrichomonas foetus and Tritrichomonas suis and treatment conditions used for preparing chromosomes Isolates
Origin
Colchicine concentrations mM (h) at 37C
Treatment periods (min) with KCl
Number of chromosomes
CAPTFa 41 T. suis CAPTF 42 T. suis CAPTF 901 T. suis CAPTF 45 T. foetus CAPTF 46 T. foetus CAPTF 165 T. foetus CAPTF 182 T. foetus CAPTF 902 T. foetus
ATCCb 30268 ATCC 30269 Czech republic ATCC 30165 ATCC 30166 Canada Canada Czech republic
0.5 (8) 0.5 (8) 0.5 (8) 0.25 (6) 0.25 (6) 0.5 (6) 0.5 (6) 0.5 (8)
90 90 90 15 15 12 15 20
5 5 5 5 5 5 5 5
a b
CAPTF: Centre for Animal Parasitology Tritrichomonas foetus Programme isolate. ATCC: American Type Culture Collection isolate.
W.-D. Xu et al. / Veterinary Parasitology 78 (1998) 247±251
249
examination of the samples at intervals during the treatment period. The suitable treatment period was identified when most of the metaphase cells were inflated to round forms and could not move on the slide. During incubation, the solution was gently shaken once or twice. Following incubation, the tubes were centrifuged at 90g for 5 min and then gently tapped to resuspend the pellets in 0.5 ml of supernatant. The cells were fixed by gradually adding a freshly prepared fixative solution (chilled methanol:acetic acid, 3:1) and constantly mixing at 48C for a minimum of 2 h. The cell suspensions were centrifuged at 90g for 5 min at 48C and the supernatant removed. The fixation step was repeated once more and the cells were resuspended by tapping the tubes. Two or three aliquots of the suspension were dropped from approximately 50 cm high onto clean, grease-free slides using plastic pipettes. After allowing the smears on the slides to dry on a slide-warmer at 458C for 1 h, the slides were stained with Diff-Quick1 (Dade Diagnostics of PR, Aguada, USA) according to the manufacturer's instructions. Slides were observed under a 40 objective lens and then photographed under oil immersion (100 objective lens). 3. Results The cells on the slides were free from overlapping, and the chromosomes were stained dark blue. Five pairs of metaphase chromosomes were observed in more than 95% of the spreads of both T. foetus (Fig. 1(A) and (B)) and T. suis (Fig. 1(C) and (D)) although aneuploids were occasionally seen on some spreads. The diploid chromosome numbers were 10 for both parasites, and all chromosomes observed had single arms. The numbers and morphology of the chromosomes of the different T. foetus and T. suis isolates appeared identical (Fig. 1(A) and (B); Table 1). However, some homologous chromosomes of T. suis appeared closely apposed to each other (Fig. 1(C) and (D)).
Fig. 1. Chromosomes of Tritrichomonas foetus isolate CAPTF 45 (A, B) and T. suis isolate CAPTF 41 (C, D) stained with Diff-Quick1 after treatment with colchicine and KCl. (2100).
250
W.-D. Xu et al. / Veterinary Parasitology 78 (1998) 247±251
4. Discussion Preparation of a flattened metaphase spread free of overlapping chromosomes is essential for determining chromosome numbers of trichomonads. Inadequate preparation may have contributed to previous reports of fewer than the actual number of chromosomes. For example, it has been reported that T. vaginalis has four chromosomes (Levine, 1961), but more recently, six have been described (Yuh et al., 1997). In the present study, it was necessary to determine and use the optimum colchicine concentrations for revealing mitotic chromosomes of T. foetus and T. suis on slides to reveal chromosome numbers and morphology. We found that Diamond's medium without agar facilitated good separation of the parasites from the medium during centrifugation. The reason for the longer hypotonic treatment (Table 1) required by T. suis is not known, but may be an indication of the osmotic difference between its cell membrane and that of T. foetus. The aneuploids were probably the result of the prolonged direct treatment of the organisms with colchicine which can cause abnormal effects on living cells. This study supports the taxonomic difference between T. vaginalis and Tritrichomonas spp. Unlike T. vaginalis (Yuh et al., 1997), the different chromosomes of both T. foetus and T. suis were similar in length. The chromosomes observed in this present study were shorter than those of T. vaginalis. The consistent numbers and morphology of chromosomes from the different isolates (Table 1) of T. foetus and T. suis are evidence of the genetic similarity among the various geographical isolates of T. foetus and T. suis. Further genetic studies are necessary to demonstrate whether or not T. foetus and T. suis are identical species. Acknowledgements Thanks to Dr. Sarah Parker for the collection of samples from bulls, the staff at the Centre for Animal Parasitology for technical support, and to Dr. M. Pakandl, Parasitology Ï eske BudeÏjovice, Czech Republic, for providing isolates Institute, Academic Science, C of T. suis and T. foetus. Financial support for this study was provided by the Canadian Food Inspection Agency's Matching Investment Initiative Program, the Prairie Farm Rehabilitation Administration of Agriculture and Agri-Food Canada, and the Saskatchewan Beef Development Board. W.D. Xu was a visiting scholar from the Veterinary Research Institute of Jilin Province, P.R. China, and was supported by the China Scholarship Council. References BonDurant, R.H., Anderson, M.L., Blanchard, P., Hird, D., Danaye-Elmi, C., Palmer, C., Sischo, W.L., Suther, D., Utterback, W., Weigler, B.J., 1990. Prevalence of trichomoniasis among California beef herds. J. Am. Vet. Med. Ass. 196, 1590±1593. Diamond, L.S., 1957. The establishment of various trichomonads of animals and man in axenic cultures. J. Parasitol. 43, 488±490.
W.-D. Xu et al. / Veterinary Parasitology 78 (1998) 247±251
251
Felleisen, R.S.J., 1997. Comparative sequence analysis of 5.8S rRNA genes and internal transcribed spacer (ITS) regions of trichomonadid protozoa. Parasitol. 155, 111±119. Fitzgerald, P.R., Johnson, A.E., Thorne, J.L., Hammond, D.M., 1955. Experimental infections of the bovine genital tract with trichomonads from swine. J. Parasitol. 41(Suppl), 17. Fitzgerald, P.R., Johnson, A.E., Hammond, D.M., Thorne, J.L., Hibler, C.P., 1958. Experimental infection of young pigs following intranasal inoculation with nasal, gastric, or cecal trichomonads from swine or with Trichomonas foetus. J. Parasitol. 44, 597±602. Hammond, D.M., Fitzgerald, P.R., 1953. Observations on trichomonads of the digestive tract and nose of pigs. J. Parasitol. 39 (suppl.), 11. Hammond, D.M., Leidl, W., 1957. Experimentelle Infecktion des Genitaltraktes beim Rind mit Trichomonaden aus dem Intestinum des Schweines. Berliner und MuÈnchener TieraÈrztliche Wochenschrift 70, 9±11. Hibler, C.P., Hammond, D.M., Caskey, F.H., Johnson, A.E., Fitzgerald, P.R., 1960. The morphology and incidence of the Trichomonads of swine, Tritrichomonas suis (Gruby and Delafond), Tritrichomonas rotunda, n. sp. and Trichomonas buttreyi, n. sp.. J. Protozool. 7, 159±171. Jensen, E.A., Hammond, D.M., 1964. A morphological study of Trichomonads and related flagellates from the bovine digestive tract. J. Protozool. 11, 386±394. Levine, N.D., 1961. Protozoan Parasites of Domestic Animals and of Man. Burgess, Minneapolis, MN, pp. 96± 98. Monk, M., 1987. Mammalian Development: A Practical Approach. IRL Press, Oxford, pp. 93±114. Speer, C.A., White, M.W., 1991. Bovine trichomoniasis. Large Animal Veterinarian 7, 18±20. Yuh, Y., Liu, J., Shaio, M., 1997. Chromosome number of Trichomonas vaginalis. J. Parasitol. 83, 551±553.
Chromosome numbers of Tritrichomonas foetus and Tritrichomonas suis Wei-Dong Xu, Zhao-Rong Lun, Alvin Gajadhar* Centre for Animal Parasitology, Canadian Food Inspection Agency, 116 Veterinary Road, Saskatoon, Saskatchewan S7N 2R3, Canada Received 30 January 1998; accepted 27 March 1998
Abstract Tritrichomonas foetus and Tritrichomonas suis isolates were cultivated axenically in Diamond's medium. Studies on the chromosome numbers of these two species with a light microscope were done by adding different concentrations of colchicine into the medium, incubating at 378C for 6±8 h and using a hypotonic swelling technique. The diploid chromosome numbers of both T. foetus and T. suis were 2n10. # 1998 Elsevier Science B.V. All rights reserved. Keywords: Chromosome; Tritrichomonas foetus; Tritrichomonas suis
1. Introduction Bovine trichomoniosis is a venereal disease of cattle associated with infertility and abortion. The etiological agent is Tritrichomonas foetus, a flagellated protozoan parasite which is the cause of significant economic losses in the free-ranging cattle industry around the world (BonDurant et al., 1990; Speer and White, 1991). Tritrichomonas suis is a closely related morphologically indistinguishable flagellated protozoan found in the nasal passages, stomach, cecum and colon of swine (Hammond and Fitzgerald, 1953; Fitzgerald et al., 1958). Tritrichomonas suis (Gruby and Delafond, 1843), and T. foetus (RiedmuÈller, 1928; Wenrich and Emmerson, 1933) were originally isolated from pigs and cattle, respectively, and given separate species names. However, the taxonomical relationship between T. suis and T. foetus needs to be resolved because T. suis isolated from pigs established infections in both heifers and bulls (Fitzgerald et al., 1955; * Corresponding author. Tel.: +1 306 9755344; fax: +1 306 9755711; e-mail: [email protected] 0304-4017/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved PII S 0 3 0 4 - 4 0 1 7 ( 9 8 ) 0 0 1 5 0 - 2
248
W.-D. Xu et al. / Veterinary Parasitology 78 (1998) 247±251
Hammond and Leidl, 1957), and characteristics of in vitro cultivation, morphology and DNA sequences between T. foetus and T. suis showed remarkable similarities (Hibler et al., 1960; Jensen and Hammond, 1964; Felleisen, 1997). Theoretically, the chromosome number in the normal genome within any species of protozoa should be constant and variation may indicate species difference. Yuh et al. (1997) reported that six chromosomes were found in Trichomonas vaginalis, the human trichomonad. However, there are no reports on the chromosome numbers of T. foetus or T. suis. The purpose of the present study was to determine the numbers of chromosomes of T. foetus and T. suis and provide an indication of their genetic relationship. 2. Materials and methods Five isolates of T. foetus and three of T. suis were used in the present study (Table 1). Both T. foetus and T. suis were axenically cultivated in Diamond's medium (Diamond, 1957) modified by omitting agar and complementing with 50 mg mlÿ1 gentamicin, 1.0 mg mlÿ1 streptomycin and 10% inactivated newborn lamb serum at 378C. Colchicine and potassium chloride (KCl) treatments were similar to the methods described by Monk (1987), and Yuh et al. (1997) with slight modifications. Prewarmed (378C) solutions of 100 mM colchicine (Sigma) were added to the media cultivating T. foetus or T. suis when the concentration of the parasites reached 1.0±1.5106 organisms per ml. A series of concentrations of colchicine (0.125, 0.25, 0.5, 1.0, 2.0, 4.0 mM) in the media were used to determine a suitable concentration for each isolate to suppress cell division by inhibiting mitosis. Trichomonads were harvested from the media at 4±8, 10, 12, 14, 16, 20, and 24 h after colchicine was added. The culture was centrifuged at 200g for 10 min at room temperature, the pellets were washed once with phosphate-buffered saline (PBS, pH 7.3, 378C), and 10 ml of prewarmed (378C) 75 mM KCl was slowly added to each tube, and the pellets gently resuspended with a pipette and incubated at 378C for 12± 90 min (Table 1). The optimal duration of KCl treatment was determined by microscopic Table 1 Isolates of Tritrichomonas foetus and Tritrichomonas suis and treatment conditions used for preparing chromosomes Isolates
Origin
Colchicine concentrations mM (h) at 37C
Treatment periods (min) with KCl
Number of chromosomes
CAPTFa 41 T. suis CAPTF 42 T. suis CAPTF 901 T. suis CAPTF 45 T. foetus CAPTF 46 T. foetus CAPTF 165 T. foetus CAPTF 182 T. foetus CAPTF 902 T. foetus
ATCCb 30268 ATCC 30269 Czech republic ATCC 30165 ATCC 30166 Canada Canada Czech republic
0.5 (8) 0.5 (8) 0.5 (8) 0.25 (6) 0.25 (6) 0.5 (6) 0.5 (6) 0.5 (8)
90 90 90 15 15 12 15 20
5 5 5 5 5 5 5 5
a b
CAPTF: Centre for Animal Parasitology Tritrichomonas foetus Programme isolate. ATCC: American Type Culture Collection isolate.
W.-D. Xu et al. / Veterinary Parasitology 78 (1998) 247±251
249
examination of the samples at intervals during the treatment period. The suitable treatment period was identified when most of the metaphase cells were inflated to round forms and could not move on the slide. During incubation, the solution was gently shaken once or twice. Following incubation, the tubes were centrifuged at 90g for 5 min and then gently tapped to resuspend the pellets in 0.5 ml of supernatant. The cells were fixed by gradually adding a freshly prepared fixative solution (chilled methanol:acetic acid, 3:1) and constantly mixing at 48C for a minimum of 2 h. The cell suspensions were centrifuged at 90g for 5 min at 48C and the supernatant removed. The fixation step was repeated once more and the cells were resuspended by tapping the tubes. Two or three aliquots of the suspension were dropped from approximately 50 cm high onto clean, grease-free slides using plastic pipettes. After allowing the smears on the slides to dry on a slide-warmer at 458C for 1 h, the slides were stained with Diff-Quick1 (Dade Diagnostics of PR, Aguada, USA) according to the manufacturer's instructions. Slides were observed under a 40 objective lens and then photographed under oil immersion (100 objective lens). 3. Results The cells on the slides were free from overlapping, and the chromosomes were stained dark blue. Five pairs of metaphase chromosomes were observed in more than 95% of the spreads of both T. foetus (Fig. 1(A) and (B)) and T. suis (Fig. 1(C) and (D)) although aneuploids were occasionally seen on some spreads. The diploid chromosome numbers were 10 for both parasites, and all chromosomes observed had single arms. The numbers and morphology of the chromosomes of the different T. foetus and T. suis isolates appeared identical (Fig. 1(A) and (B); Table 1). However, some homologous chromosomes of T. suis appeared closely apposed to each other (Fig. 1(C) and (D)).
Fig. 1. Chromosomes of Tritrichomonas foetus isolate CAPTF 45 (A, B) and T. suis isolate CAPTF 41 (C, D) stained with Diff-Quick1 after treatment with colchicine and KCl. (2100).
250
W.-D. Xu et al. / Veterinary Parasitology 78 (1998) 247±251
4. Discussion Preparation of a flattened metaphase spread free of overlapping chromosomes is essential for determining chromosome numbers of trichomonads. Inadequate preparation may have contributed to previous reports of fewer than the actual number of chromosomes. For example, it has been reported that T. vaginalis has four chromosomes (Levine, 1961), but more recently, six have been described (Yuh et al., 1997). In the present study, it was necessary to determine and use the optimum colchicine concentrations for revealing mitotic chromosomes of T. foetus and T. suis on slides to reveal chromosome numbers and morphology. We found that Diamond's medium without agar facilitated good separation of the parasites from the medium during centrifugation. The reason for the longer hypotonic treatment (Table 1) required by T. suis is not known, but may be an indication of the osmotic difference between its cell membrane and that of T. foetus. The aneuploids were probably the result of the prolonged direct treatment of the organisms with colchicine which can cause abnormal effects on living cells. This study supports the taxonomic difference between T. vaginalis and Tritrichomonas spp. Unlike T. vaginalis (Yuh et al., 1997), the different chromosomes of both T. foetus and T. suis were similar in length. The chromosomes observed in this present study were shorter than those of T. vaginalis. The consistent numbers and morphology of chromosomes from the different isolates (Table 1) of T. foetus and T. suis are evidence of the genetic similarity among the various geographical isolates of T. foetus and T. suis. Further genetic studies are necessary to demonstrate whether or not T. foetus and T. suis are identical species. Acknowledgements Thanks to Dr. Sarah Parker for the collection of samples from bulls, the staff at the Centre for Animal Parasitology for technical support, and to Dr. M. Pakandl, Parasitology Ï eske BudeÏjovice, Czech Republic, for providing isolates Institute, Academic Science, C of T. suis and T. foetus. Financial support for this study was provided by the Canadian Food Inspection Agency's Matching Investment Initiative Program, the Prairie Farm Rehabilitation Administration of Agriculture and Agri-Food Canada, and the Saskatchewan Beef Development Board. W.D. Xu was a visiting scholar from the Veterinary Research Institute of Jilin Province, P.R. China, and was supported by the China Scholarship Council. References BonDurant, R.H., Anderson, M.L., Blanchard, P., Hird, D., Danaye-Elmi, C., Palmer, C., Sischo, W.L., Suther, D., Utterback, W., Weigler, B.J., 1990. Prevalence of trichomoniasis among California beef herds. J. Am. Vet. Med. Ass. 196, 1590±1593. Diamond, L.S., 1957. The establishment of various trichomonads of animals and man in axenic cultures. J. Parasitol. 43, 488±490.
W.-D. Xu et al. / Veterinary Parasitology 78 (1998) 247±251
251
Felleisen, R.S.J., 1997. Comparative sequence analysis of 5.8S rRNA genes and internal transcribed spacer (ITS) regions of trichomonadid protozoa. Parasitol. 155, 111±119. Fitzgerald, P.R., Johnson, A.E., Thorne, J.L., Hammond, D.M., 1955. Experimental infections of the bovine genital tract with trichomonads from swine. J. Parasitol. 41(Suppl), 17. Fitzgerald, P.R., Johnson, A.E., Hammond, D.M., Thorne, J.L., Hibler, C.P., 1958. Experimental infection of young pigs following intranasal inoculation with nasal, gastric, or cecal trichomonads from swine or with Trichomonas foetus. J. Parasitol. 44, 597±602. Hammond, D.M., Fitzgerald, P.R., 1953. Observations on trichomonads of the digestive tract and nose of pigs. J. Parasitol. 39 (suppl.), 11. Hammond, D.M., Leidl, W., 1957. Experimentelle Infecktion des Genitaltraktes beim Rind mit Trichomonaden aus dem Intestinum des Schweines. Berliner und MuÈnchener TieraÈrztliche Wochenschrift 70, 9±11. Hibler, C.P., Hammond, D.M., Caskey, F.H., Johnson, A.E., Fitzgerald, P.R., 1960. The morphology and incidence of the Trichomonads of swine, Tritrichomonas suis (Gruby and Delafond), Tritrichomonas rotunda, n. sp. and Trichomonas buttreyi, n. sp.. J. Protozool. 7, 159±171. Jensen, E.A., Hammond, D.M., 1964. A morphological study of Trichomonads and related flagellates from the bovine digestive tract. J. Protozool. 11, 386±394. Levine, N.D., 1961. Protozoan Parasites of Domestic Animals and of Man. Burgess, Minneapolis, MN, pp. 96± 98. Monk, M., 1987. Mammalian Development: A Practical Approach. IRL Press, Oxford, pp. 93±114. Speer, C.A., White, M.W., 1991. Bovine trichomoniasis. Large Animal Veterinarian 7, 18±20. Yuh, Y., Liu, J., Shaio, M., 1997. Chromosome number of Trichomonas vaginalis. J. Parasitol. 83, 551±553.