- Email: [email protected]
Experimental Cochlosoma anatis infections in poultry
Veterinary Parasitology 81 (1999) 21±27
Experimental Cochlosoma anatis infections in poultry David S. Lindsaya,*, Calvert T. Larsenb, Anne M. Zajaca, F. William Piersonb a
Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, 1410 Prices Fork Road, Blacksburg, VA 24061-0342, USA b Center for Molecular Medicine and Infectious Diseases, Department of Large Animal Clinical Sciences, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, 1410 Prices Fork Road, Blacksburg, VA 24061-0342, USA Received 4 June 1998; accepted 8 September 1998
Abstract Cochlosoma anatis [KotlaÂn, A., 1923. Zentralbl. Bakteriol. Parasitenkd. Infektienskr. Hyg. 90, pp. 24±28] is a flagellated protozoan parasite of birds. We have encountered C. anatis in turkeys with enteritis. Experimental oral inoculations of turkeys with 1 106 to 10 106 trophozoites consistently reproduced infections in recipients. Trophozoites were most numerous in the jejunum and ileum but could be observed in the duodenum, ceca, colon, and feces. When 12 naive turkeys were placed on contaminated litter vacated by excreting turkeys only one of 12 became infected. When eight naive turkeys were placed in boxes with birds currently excreting trophozoites, seven of eight became infected. Trophozoites could not survive exposure to water or to freezing. Attempts to culture trophozoites in modified Diamond's medium, Kiester's medium, RPMI 1640 medium with 10% fetal bovine serum, or on cultured bovine turbinate cells were not successful. Four of six bobwhite quail and one of eight chickens orally inoculated with 10 106 to 20 106 trophozoites had detectable infections. Trophozoites were observed only in the ilea of bobwhite quail and the ceca of the positive chicken. Trophozoites collected from chickens and bobwhite quail remained infectious for turkeys. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Turkey; Chicken; Quail; Cochlosoma anatis; Flagellates; Protozoan
1. Introduction Cochlosoma anatis (KotlaÂn, 1923) is a rarely studied flagellated protozoan parasite of birds (Pecka et al., 1996). It was originally described from the intestines of young ducks * Corresponding author. Tel.: +1-540-231-6302; fax: +1-540-231-3426; e-mail: [email protected] 0304-4017/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 9 8 ) 0 0 2 2 7 - 1
22
D.S. Lindsay et al. / Veterinary Parasitology 81 (1999) 21±27
with coccidiosis (KotlaÂn, 1923). The trophozoites are uninucleate and resemble Giardia lamblia in that they have a ventral sucking disk. Cochlosoma anatis has no cyst stage and it is related to trichomonads (Pecka et al., 1996). It has recently been associated with runting in ducklings (Bollinger and Barker, 1996; Bollinger et al., 1996) and enteritis in turkeys (Cooper et al., 1995). Nothing is known about the transmission or host specificity of this parasite in poultry. The present studies were done to determine factors involved in the transmission of the parasite, to determine where it develops in young turkeys (Meleagris gallopavo), and to determine if trophozoites isolated from turkeys were infectious for bobwhite quail (Colinus virginianus) or chickens (Gallus gallus).
2. Materials and methods 2.1. Cochlosoma anatis isolate and its maintenance Trophozoites were originally isolated from the intestine of a naturally infected turkey from northern Virginia, USA. Trophozoites were maintained by oral subinoculation of 1 106 to 10 106 trophozoites into naive turkeys. For experimental inoculations, donor turkeys were killed and their small intestines opened, the mucosa was gently scraped with a 22 22 mm2 coverslip and the scrapings placed in 30 to 40 ml of Hanks' balanced salt solution without calcium and magnesium (HBSS). This mixture was filtered through two layers of cheese cloth to remove particulate matter. The numbers of trophozoites present were determined by mixing 100 ml of trophozoite mixture with 100 ml of 10% neutral buffered formalin solution and counting the immobilized trophozoites in a hemocytometer. In vitro cultivation of C. anatis trophozoites was attempted in modified Diamond's medium, Kiester's medium, and RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum and 100 u mlÿ1 penicillin plus 100 mg mlÿ1 streptomycin. Cell culture monolayers of bovine turbinate cells were also inoculated with C. anatis trophozoites and examined for growth. 2.2. Bird maintenance Birds were kept in groups of two to five in 30.5 cm(wide) 61 cm(long) 50 cm(tall) cardboard boxes with wood shavings as litter and a wire mesh top. Water and nonmedicated commercial poultry rations were provided ad libitum. 2.3. Factors influencing trophozoite survival Freshly collected trophozoites were placed in tap water, distilled water, frozen in HBSS at ÿ208C or kept at 378C in HBSS for 45 min then examined for motility. Other freshly collected trophozoites samples were kept in HBSS at 48C, room temperature
D.S. Lindsay et al. / Veterinary Parasitology 81 (1999) 21±27
23
(22±258C), or at 378C for 24 h then examined for motility. An in vivo study was done in which freshly collected trophozoites were placed in tap water for 5 min then spun down by centrifugation and placed in HBSS. Three turkeys were orally inoculated with 1 107 water treated trophozoites. 2.4. Cochlosoma anatis infection dynamics in turkeys Twenty, 30 day-old turkeys were orally inoculated with 1 107 trophozoites and divided into five groups of four turkeys per box. One turkey was removed from each box and examined at necropsy on days 4, 5, 6, and 7 postinoculation (PI). At necropsy, the intestinal tract was removed and opened from the duodenum to the cloaca including ceca. Smears were made from the duodenum, jejunum, ileum, cecal neck, and colon of each bird using a 22 22 mm2 coverslip and placed on a glass microscope slide for examination using Normarski interference contrast microscopy. Additionally, a 4 cm portion of jejunum (2 cm on each side of Meckel's diverticulum) was placed in 10 ml of HBSS in a 15 ml conical centrifuge tube, mixed and the numbers of trophozoites present were determined by mixing 100 ml of trophozoite mixture with 100 ml of 10% neutral buffered formalin solution and counting the immobilized trophozoites in a hemocytometer. 2.5. Scanning electron microscopy Concentrated trophozoites were fixed as a suspension in 3% glutaraldehyde and processed for scanning electron microscopy. Samples were examined in a JOEL 358C scanning electron microscope operating at 15 kV. 2.6. Cross species transmission Eight, 8 week-old chickens were orally inoculated with 2 107 trophozoites and six adult bobwhite quail were orally inoculated with 2 107 trophozoites (two birds) or 1 107 trophozoites (four birds). The birds were killed and examined at necropsy 11±13 days PI. Trophozoites from positive bobwhite quail or chickens were collected and subinoculated into turkeys. 2.7. Litter transmission Twelve, 16 day-old turkeys were placed in boxes that had been used to house turkeys excreting C. anatis trophozoites for at least 2 days. The naive turkeys were placed on the contaminated litter less than 2 h after the donor birds were removed. They were killed and examined at necropsy examined 8 PI (seven turkeys), 11 PI (three turkeys), or 14 PI (two turkeys). Another study was done in which eight, 7 day-old naive turkeys were placed in a box with a turkey that was infected with C. anatis. These turkeys were then killed 7 days PI and examined at necropsy.
24
D.S. Lindsay et al. / Veterinary Parasitology 81 (1999) 21±27
3. Results 3.1. Cochlosoma anatis isolate maintenance Our isolate has been maintained in experimentally infected turkeys since December 5, 1997 without any obvious loss of infectivity. Our attempts to obtain growth of the parasite in defined media have been unsuccessful. 3.2. Factors influencing trophozoite survival Trophozoites exposed to tap or distilled water for 45 min at 378C or kept at ÿ208C in HBSS were nonmotile and appeared dead upon microscopic examination. Trophozoites kept at 378C for 45 min in HBSS were motile and appeared viable. Trophozoites kept in HBSS at 48C, room temperature or 378C for 24 h were nonmotile and appeared dead. None of the turkeys inoculated with trophozoites exposed to water for 5 min had demonstrable infections when examined at necropsy. 3.3. Infection dynamics in turkeys Two of five turkeys examined 4 days PI were positive for trophozoites. Trophozoites were present only in the duodenum of one turkey and only in the jejunum of the other positive turkey. The mean trophozoite count 4 day PI was 2 103 (range, 0.0 104 to 1 104; SD 4.5 103). Four of five turkeys examined 5 days PI were positive. Trophozoites were present in the duodenum, jejunum, and ileum of all positive turkeys and in the ceca of three and colon of two turkeys. The mean trophozoite count 5 days PI was 2.01 106 (range 0.0 106 to 7.24 106; SD 2.97 106). All five turkeys examined 6 days PI were positive for trophozoites. Three of the five turkeys had trophozoites in all intestinal locations examined. One turkey had trophozoites confined to the jejunum and ileum while the other turkey had trophozoites in all regions except the ceca. The mean trophozoite count 6 days PI was 4.19 106 (range 6.0 104 to 8.08 106; SD 3.53 106). All five turkeys examined 7 days PI were positive for trophozoites. Four of the five turkeys had trophozoites in all intestinal locations examined. The other turkey had trophozoites in all regions except the colon. The mean trophozoite count 7 days PI was 5.03 106 (range 2.4 104 to 7.46 106; SD 1.81 106). 3.4. Scanning electron microscopy Examination of trophozoite preparations with SEM demonstrated numerous flagellate protozoa with six flagellae, an undulating membrane, prominent lateral grove and ventral sucking disk (Figs. 1 and 2). Four anterior flagellae and a recurrent flagella adherent to the undulating membrane arose from the ventral sucking disk and the sixth flagella emerged from the dorsal surface of the trophozoite. An axostyle was occasionally visible on some trophozoites.
D.S. Lindsay et al. / Veterinary Parasitology 81 (1999) 21±27
25
Fig. 1. Scanning electron micrograph of the dorsal surface of two Cochlosoma anatis trophozoites. Note the dorsal flagellum (arrowhead) and anteriorly emerging flagella (arrow) of each trophozoite. An axostyle (curved arrow) is visible on one of the trophozoite. 6660. Bar 5 mm.
3.5. Cross species transmission Four of six bobwhite quail and one of eight chickens orally inoculated with 10 106 to 20 106 trophozoites had demonstrable infections. Trophozoites were observed only in the ilea of bobwhite quail and the ceca of the positive chicken. Trophozoites collected from chickens and bobwhite quail remained infectious for turkeys. 3.6. Litter transmission When 12 naive turkeys were placed on contaminated litter vacated by turkeys with an active infection of C. anatis, none of the seven examined 8 days PI, one of three examined 11 days PI, and none of the two examined 14 days PI were positive for
Fig. 2. Scanning electron micrograph of the ventral surface of a Cochlosoma anatis trophozoite. Note the ventral sucking disk (S), the prominent grove, (G), anterior flagella (arrow), undulating membrane (double arrowhead) and recurrent flagellum (arrowhead). 6660. Bar 5 mm.
26
D.S. Lindsay et al. / Veterinary Parasitology 81 (1999) 21±27
C. anatis. When eight naive turkeys were placed in boxes with birds currently excreting trophozoites seven of eight were infected by 7 days PI. 4. Discussion Our study demonstrated that Cochlosoma anatis was orally infective for turkeys and that development occurred throughout the digestive tract. The trophozoites needed 5 to 6 days to reach numbers detectable in most turkeys orally inoculated with 1 107 trophozoites. Our study was not designed to determine if C. anatis was a significant pathogen of turkeys, however natural cases of enteritis associated with C. anatis infection have been reported in turkeys (McNeil and Hinshaw, 1942; Campbell, 1945; Cooper et al., 1995). Further studies are needed to define the role of C. anatis as a primary or secondary pathogen in turkeys. Trophozoites of C. anatis were readily destroyed by immersion in water. This indicates that contamination of feed or litter would be the major source of infection for other turkeys. Only one of 12 turkeys became infected when placed on litter contaminated by experimentally infected birds when the trophozoite shedding turkeys had been removed. This suggests a lack of long-term survival of trophozoites in the environment. The greater success of transmission (seven of eight turkeys) when naive turkeys were placed in boxes with turkeys that were excreting trophozoites also indicates that trophozoites need to be acquired soon after excretion into the environment. Our SEM findings are similar to those reported in naturally infected turkeys (Cooper et al., 1995) and geese (Pecka et al., 1996). Cooper et al. (1995) did not describe an undulating membrane in trophozoites but one is present in trophozoites in their micrographs. Cochlosoma sp. have not previously been reported in bobwhite quail or chickens. Cochlosoma anatis or C. anatis-like protozoa have been observed in naturally infected water fowl including white Peking ducks (Anas platyrhynchos), mallards (An. platyrhynchos), Muscovy ducks (Cairina moschata), pintails (An. acuta), lesser scaups (Aythya affinis), golden eye ducklings (Bucephela clangula), buffleheads (B. albeola), cinnamon teal (An. cyanoptera), marbled teal (An. angustirostris), Chilean pintails (An. georgia georgia), South African yellow-billed ducks (An. undulata), ring-necked ducks (Ay. collaris), coots (Fulica atra) and domestic geese (Anser anser) (Travis, 1938; Pecka, 1991; Pecka et al., 1996; Bollinger and Barker, 1996). Filippich and O'Donoghue (1997) reported finding Cochlosoma sp. in the feces of eight breeds of finches. Reports of Cochlosoma sp. in bats and shrews (Watkins et al., 1989; Pecka et al., 1996) indicate that this genus is not restricted to avian hosts. References Bollinger, T.K., Barker, I.K., 1996. Runting of ducklings associated with Cochlosoma anatis infection. Avian Dis. 40, 181±185. Bollinger, T.K., Barker, I.K., Fernando, M.A., 1996. Effects of the intestinal flagellate, Cochlosoma anatis, on intestinal mucosal morphology and disaccharidase activity in Muscovy ducklings. Int. J. Parasitol. 26, 533±542.
D.S. Lindsay et al. / Veterinary Parasitology 81 (1999) 21±27
27
Campbell, J.G., 1945. An infectious enteritis of young turkeys associated with Cochlosoma sp. Vet. J. 101, 255±259. Cooper, G.L., Shivaprasad, H.L., Bickford, A.A., Nordhausen, R., Munn, R.J., Jeffrey, J.S., 1995. Enteritis in turkeys associated with an unusual flagellated protozoan (Cochlosoma anatis). Avian Dis. 39, 183±190. Filippich, L.J., O'Donoghue, P.J., 1997. Cochlosoma infections in finches. Aust. Vet. J. 75, 561±563. KotlaÂn, A., 1923. Zur Kenntnis der Darmflagellaten aus der Hausente und anderen WasservoÈgeln. Zentralbl. Bakteriol. Parasitenkd. Infektienskr. Hyg. 90, 24±28. McNeil, E., Hinshaw, W.R., 1942. Cochlosoma rostratum from the turkey. J. Parasitol. 28, 349±350. Pecka, Z., 1991. Domestic geese (Anser anser L.) as a new host of Cochlosoma anatis. Folia Parasitol. 38, 91±92. Pecka, Z., NohynkovaÂ, E., Kulda, J., 1996. Ultrastructure of Cochlosoma anatis KotlaÂn, 1923 and taxonomic position of the family Cochlosomatidae (Parabasala: Trichomonadia). Euorp. J. Protistol. 32, 190±201. Travis, B.V., 1938. A synopsis of the flagellate genus Cochlosoma KotlaÂn, with a description of two new species. J. Parasitol. 24, 343±351. Watkins, R.A., O'Dell, W.D., Pinter, A.J., 1989. Redescription of flagellar arrangement in the duck intestinal flagellate, Cochlosoma anatis and description of a new species, Cochlosoma soricis n. sp. from shrews. J. Protozool. 36, 527±531.
Experimental Cochlosoma anatis infections in poultry David S. Lindsaya,*, Calvert T. Larsenb, Anne M. Zajaca, F. William Piersonb a
Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, 1410 Prices Fork Road, Blacksburg, VA 24061-0342, USA b Center for Molecular Medicine and Infectious Diseases, Department of Large Animal Clinical Sciences, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, 1410 Prices Fork Road, Blacksburg, VA 24061-0342, USA Received 4 June 1998; accepted 8 September 1998
Abstract Cochlosoma anatis [KotlaÂn, A., 1923. Zentralbl. Bakteriol. Parasitenkd. Infektienskr. Hyg. 90, pp. 24±28] is a flagellated protozoan parasite of birds. We have encountered C. anatis in turkeys with enteritis. Experimental oral inoculations of turkeys with 1 106 to 10 106 trophozoites consistently reproduced infections in recipients. Trophozoites were most numerous in the jejunum and ileum but could be observed in the duodenum, ceca, colon, and feces. When 12 naive turkeys were placed on contaminated litter vacated by excreting turkeys only one of 12 became infected. When eight naive turkeys were placed in boxes with birds currently excreting trophozoites, seven of eight became infected. Trophozoites could not survive exposure to water or to freezing. Attempts to culture trophozoites in modified Diamond's medium, Kiester's medium, RPMI 1640 medium with 10% fetal bovine serum, or on cultured bovine turbinate cells were not successful. Four of six bobwhite quail and one of eight chickens orally inoculated with 10 106 to 20 106 trophozoites had detectable infections. Trophozoites were observed only in the ilea of bobwhite quail and the ceca of the positive chicken. Trophozoites collected from chickens and bobwhite quail remained infectious for turkeys. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Turkey; Chicken; Quail; Cochlosoma anatis; Flagellates; Protozoan
1. Introduction Cochlosoma anatis (KotlaÂn, 1923) is a rarely studied flagellated protozoan parasite of birds (Pecka et al., 1996). It was originally described from the intestines of young ducks * Corresponding author. Tel.: +1-540-231-6302; fax: +1-540-231-3426; e-mail: [email protected] 0304-4017/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 9 8 ) 0 0 2 2 7 - 1
22
D.S. Lindsay et al. / Veterinary Parasitology 81 (1999) 21±27
with coccidiosis (KotlaÂn, 1923). The trophozoites are uninucleate and resemble Giardia lamblia in that they have a ventral sucking disk. Cochlosoma anatis has no cyst stage and it is related to trichomonads (Pecka et al., 1996). It has recently been associated with runting in ducklings (Bollinger and Barker, 1996; Bollinger et al., 1996) and enteritis in turkeys (Cooper et al., 1995). Nothing is known about the transmission or host specificity of this parasite in poultry. The present studies were done to determine factors involved in the transmission of the parasite, to determine where it develops in young turkeys (Meleagris gallopavo), and to determine if trophozoites isolated from turkeys were infectious for bobwhite quail (Colinus virginianus) or chickens (Gallus gallus).
2. Materials and methods 2.1. Cochlosoma anatis isolate and its maintenance Trophozoites were originally isolated from the intestine of a naturally infected turkey from northern Virginia, USA. Trophozoites were maintained by oral subinoculation of 1 106 to 10 106 trophozoites into naive turkeys. For experimental inoculations, donor turkeys were killed and their small intestines opened, the mucosa was gently scraped with a 22 22 mm2 coverslip and the scrapings placed in 30 to 40 ml of Hanks' balanced salt solution without calcium and magnesium (HBSS). This mixture was filtered through two layers of cheese cloth to remove particulate matter. The numbers of trophozoites present were determined by mixing 100 ml of trophozoite mixture with 100 ml of 10% neutral buffered formalin solution and counting the immobilized trophozoites in a hemocytometer. In vitro cultivation of C. anatis trophozoites was attempted in modified Diamond's medium, Kiester's medium, and RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum and 100 u mlÿ1 penicillin plus 100 mg mlÿ1 streptomycin. Cell culture monolayers of bovine turbinate cells were also inoculated with C. anatis trophozoites and examined for growth. 2.2. Bird maintenance Birds were kept in groups of two to five in 30.5 cm(wide) 61 cm(long) 50 cm(tall) cardboard boxes with wood shavings as litter and a wire mesh top. Water and nonmedicated commercial poultry rations were provided ad libitum. 2.3. Factors influencing trophozoite survival Freshly collected trophozoites were placed in tap water, distilled water, frozen in HBSS at ÿ208C or kept at 378C in HBSS for 45 min then examined for motility. Other freshly collected trophozoites samples were kept in HBSS at 48C, room temperature
D.S. Lindsay et al. / Veterinary Parasitology 81 (1999) 21±27
23
(22±258C), or at 378C for 24 h then examined for motility. An in vivo study was done in which freshly collected trophozoites were placed in tap water for 5 min then spun down by centrifugation and placed in HBSS. Three turkeys were orally inoculated with 1 107 water treated trophozoites. 2.4. Cochlosoma anatis infection dynamics in turkeys Twenty, 30 day-old turkeys were orally inoculated with 1 107 trophozoites and divided into five groups of four turkeys per box. One turkey was removed from each box and examined at necropsy on days 4, 5, 6, and 7 postinoculation (PI). At necropsy, the intestinal tract was removed and opened from the duodenum to the cloaca including ceca. Smears were made from the duodenum, jejunum, ileum, cecal neck, and colon of each bird using a 22 22 mm2 coverslip and placed on a glass microscope slide for examination using Normarski interference contrast microscopy. Additionally, a 4 cm portion of jejunum (2 cm on each side of Meckel's diverticulum) was placed in 10 ml of HBSS in a 15 ml conical centrifuge tube, mixed and the numbers of trophozoites present were determined by mixing 100 ml of trophozoite mixture with 100 ml of 10% neutral buffered formalin solution and counting the immobilized trophozoites in a hemocytometer. 2.5. Scanning electron microscopy Concentrated trophozoites were fixed as a suspension in 3% glutaraldehyde and processed for scanning electron microscopy. Samples were examined in a JOEL 358C scanning electron microscope operating at 15 kV. 2.6. Cross species transmission Eight, 8 week-old chickens were orally inoculated with 2 107 trophozoites and six adult bobwhite quail were orally inoculated with 2 107 trophozoites (two birds) or 1 107 trophozoites (four birds). The birds were killed and examined at necropsy 11±13 days PI. Trophozoites from positive bobwhite quail or chickens were collected and subinoculated into turkeys. 2.7. Litter transmission Twelve, 16 day-old turkeys were placed in boxes that had been used to house turkeys excreting C. anatis trophozoites for at least 2 days. The naive turkeys were placed on the contaminated litter less than 2 h after the donor birds were removed. They were killed and examined at necropsy examined 8 PI (seven turkeys), 11 PI (three turkeys), or 14 PI (two turkeys). Another study was done in which eight, 7 day-old naive turkeys were placed in a box with a turkey that was infected with C. anatis. These turkeys were then killed 7 days PI and examined at necropsy.
24
D.S. Lindsay et al. / Veterinary Parasitology 81 (1999) 21±27
3. Results 3.1. Cochlosoma anatis isolate maintenance Our isolate has been maintained in experimentally infected turkeys since December 5, 1997 without any obvious loss of infectivity. Our attempts to obtain growth of the parasite in defined media have been unsuccessful. 3.2. Factors influencing trophozoite survival Trophozoites exposed to tap or distilled water for 45 min at 378C or kept at ÿ208C in HBSS were nonmotile and appeared dead upon microscopic examination. Trophozoites kept at 378C for 45 min in HBSS were motile and appeared viable. Trophozoites kept in HBSS at 48C, room temperature or 378C for 24 h were nonmotile and appeared dead. None of the turkeys inoculated with trophozoites exposed to water for 5 min had demonstrable infections when examined at necropsy. 3.3. Infection dynamics in turkeys Two of five turkeys examined 4 days PI were positive for trophozoites. Trophozoites were present only in the duodenum of one turkey and only in the jejunum of the other positive turkey. The mean trophozoite count 4 day PI was 2 103 (range, 0.0 104 to 1 104; SD 4.5 103). Four of five turkeys examined 5 days PI were positive. Trophozoites were present in the duodenum, jejunum, and ileum of all positive turkeys and in the ceca of three and colon of two turkeys. The mean trophozoite count 5 days PI was 2.01 106 (range 0.0 106 to 7.24 106; SD 2.97 106). All five turkeys examined 6 days PI were positive for trophozoites. Three of the five turkeys had trophozoites in all intestinal locations examined. One turkey had trophozoites confined to the jejunum and ileum while the other turkey had trophozoites in all regions except the ceca. The mean trophozoite count 6 days PI was 4.19 106 (range 6.0 104 to 8.08 106; SD 3.53 106). All five turkeys examined 7 days PI were positive for trophozoites. Four of the five turkeys had trophozoites in all intestinal locations examined. The other turkey had trophozoites in all regions except the colon. The mean trophozoite count 7 days PI was 5.03 106 (range 2.4 104 to 7.46 106; SD 1.81 106). 3.4. Scanning electron microscopy Examination of trophozoite preparations with SEM demonstrated numerous flagellate protozoa with six flagellae, an undulating membrane, prominent lateral grove and ventral sucking disk (Figs. 1 and 2). Four anterior flagellae and a recurrent flagella adherent to the undulating membrane arose from the ventral sucking disk and the sixth flagella emerged from the dorsal surface of the trophozoite. An axostyle was occasionally visible on some trophozoites.
D.S. Lindsay et al. / Veterinary Parasitology 81 (1999) 21±27
25
Fig. 1. Scanning electron micrograph of the dorsal surface of two Cochlosoma anatis trophozoites. Note the dorsal flagellum (arrowhead) and anteriorly emerging flagella (arrow) of each trophozoite. An axostyle (curved arrow) is visible on one of the trophozoite. 6660. Bar 5 mm.
3.5. Cross species transmission Four of six bobwhite quail and one of eight chickens orally inoculated with 10 106 to 20 106 trophozoites had demonstrable infections. Trophozoites were observed only in the ilea of bobwhite quail and the ceca of the positive chicken. Trophozoites collected from chickens and bobwhite quail remained infectious for turkeys. 3.6. Litter transmission When 12 naive turkeys were placed on contaminated litter vacated by turkeys with an active infection of C. anatis, none of the seven examined 8 days PI, one of three examined 11 days PI, and none of the two examined 14 days PI were positive for
Fig. 2. Scanning electron micrograph of the ventral surface of a Cochlosoma anatis trophozoite. Note the ventral sucking disk (S), the prominent grove, (G), anterior flagella (arrow), undulating membrane (double arrowhead) and recurrent flagellum (arrowhead). 6660. Bar 5 mm.
26
D.S. Lindsay et al. / Veterinary Parasitology 81 (1999) 21±27
C. anatis. When eight naive turkeys were placed in boxes with birds currently excreting trophozoites seven of eight were infected by 7 days PI. 4. Discussion Our study demonstrated that Cochlosoma anatis was orally infective for turkeys and that development occurred throughout the digestive tract. The trophozoites needed 5 to 6 days to reach numbers detectable in most turkeys orally inoculated with 1 107 trophozoites. Our study was not designed to determine if C. anatis was a significant pathogen of turkeys, however natural cases of enteritis associated with C. anatis infection have been reported in turkeys (McNeil and Hinshaw, 1942; Campbell, 1945; Cooper et al., 1995). Further studies are needed to define the role of C. anatis as a primary or secondary pathogen in turkeys. Trophozoites of C. anatis were readily destroyed by immersion in water. This indicates that contamination of feed or litter would be the major source of infection for other turkeys. Only one of 12 turkeys became infected when placed on litter contaminated by experimentally infected birds when the trophozoite shedding turkeys had been removed. This suggests a lack of long-term survival of trophozoites in the environment. The greater success of transmission (seven of eight turkeys) when naive turkeys were placed in boxes with turkeys that were excreting trophozoites also indicates that trophozoites need to be acquired soon after excretion into the environment. Our SEM findings are similar to those reported in naturally infected turkeys (Cooper et al., 1995) and geese (Pecka et al., 1996). Cooper et al. (1995) did not describe an undulating membrane in trophozoites but one is present in trophozoites in their micrographs. Cochlosoma sp. have not previously been reported in bobwhite quail or chickens. Cochlosoma anatis or C. anatis-like protozoa have been observed in naturally infected water fowl including white Peking ducks (Anas platyrhynchos), mallards (An. platyrhynchos), Muscovy ducks (Cairina moschata), pintails (An. acuta), lesser scaups (Aythya affinis), golden eye ducklings (Bucephela clangula), buffleheads (B. albeola), cinnamon teal (An. cyanoptera), marbled teal (An. angustirostris), Chilean pintails (An. georgia georgia), South African yellow-billed ducks (An. undulata), ring-necked ducks (Ay. collaris), coots (Fulica atra) and domestic geese (Anser anser) (Travis, 1938; Pecka, 1991; Pecka et al., 1996; Bollinger and Barker, 1996). Filippich and O'Donoghue (1997) reported finding Cochlosoma sp. in the feces of eight breeds of finches. Reports of Cochlosoma sp. in bats and shrews (Watkins et al., 1989; Pecka et al., 1996) indicate that this genus is not restricted to avian hosts. References Bollinger, T.K., Barker, I.K., 1996. Runting of ducklings associated with Cochlosoma anatis infection. Avian Dis. 40, 181±185. Bollinger, T.K., Barker, I.K., Fernando, M.A., 1996. Effects of the intestinal flagellate, Cochlosoma anatis, on intestinal mucosal morphology and disaccharidase activity in Muscovy ducklings. Int. J. Parasitol. 26, 533±542.
D.S. Lindsay et al. / Veterinary Parasitology 81 (1999) 21±27
27
Campbell, J.G., 1945. An infectious enteritis of young turkeys associated with Cochlosoma sp. Vet. J. 101, 255±259. Cooper, G.L., Shivaprasad, H.L., Bickford, A.A., Nordhausen, R., Munn, R.J., Jeffrey, J.S., 1995. Enteritis in turkeys associated with an unusual flagellated protozoan (Cochlosoma anatis). Avian Dis. 39, 183±190. Filippich, L.J., O'Donoghue, P.J., 1997. Cochlosoma infections in finches. Aust. Vet. J. 75, 561±563. KotlaÂn, A., 1923. Zur Kenntnis der Darmflagellaten aus der Hausente und anderen WasservoÈgeln. Zentralbl. Bakteriol. Parasitenkd. Infektienskr. Hyg. 90, 24±28. McNeil, E., Hinshaw, W.R., 1942. Cochlosoma rostratum from the turkey. J. Parasitol. 28, 349±350. Pecka, Z., 1991. Domestic geese (Anser anser L.) as a new host of Cochlosoma anatis. Folia Parasitol. 38, 91±92. Pecka, Z., NohynkovaÂ, E., Kulda, J., 1996. Ultrastructure of Cochlosoma anatis KotlaÂn, 1923 and taxonomic position of the family Cochlosomatidae (Parabasala: Trichomonadia). Euorp. J. Protistol. 32, 190±201. Travis, B.V., 1938. A synopsis of the flagellate genus Cochlosoma KotlaÂn, with a description of two new species. J. Parasitol. 24, 343±351. Watkins, R.A., O'Dell, W.D., Pinter, A.J., 1989. Redescription of flagellar arrangement in the duck intestinal flagellate, Cochlosoma anatis and description of a new species, Cochlosoma soricis n. sp. from shrews. J. Protozool. 36, 527±531.