J. PROTOZOOL. 2 3 ( 1 ) , 155-158 (1976).
Eimeria tenella: Host Specificity in Gallinaceous Birds J. M. VETTERLING United States Department of Agriculture, Animal Parasitology Institute, Agricultural Research Service, Beltsville, Maryland 20705
SYNOPSIS. Eight species representing 8 genera of gallinaceous birds were used: Alectoris graeca; Colinus virginianus; Coturnix coturnix; Callus gallus; Meleagris gallopavo ; Numidia meleagris; Pavo cristatus; Phasianus colchicus. Threeweek-old birds were dosed with sporulated oocysts of Eimeria tenella Beltsville strain. At 4, 24, 48, 72, 96, 120, 144, and 168 hr after inoculation, 1-3 infected birds and uninoculated controls of each species were killed by cardiac exsanguination. Pieces of intestines were fixed and examined for stages of E. tenella as stained paraffin sections or indirect fluorescent antibody preparations. Oocyst counts were made in droppings collected for the first 6 days of the patent period. Sporozoites were found in the lamina propria of some birds of 5 species at 4 hr postinoculation, but no stages were found thereafter except in the breeds of G . gallus and A . graeca. At 144 and 168 hr postinoculation, a few macrogametes were found in the ceca of 2 A . graeca, but no oocysts were found in the feces. No statistical difference was found between the number of oocysts produced/bird in the breeds of G. gallus examined. It is evident from these observations that E . tenella did not complete its life cycle in several close phylogenetic relatives of G. gallus, even though in other studies this parasite was found to complete its life cycle in cell cultures derived from the same birds. Index Key Words: Eimeria tenella; host specificity; Galliformes; Alectoris graeca; Colinus virginianus; Coturnix coturnix; Gallus gallus; Meleagris gallopavo; Numidia meleagris; Pavo cristatus; Phasianus colchicus.
A
TTEMPTS to transmit Eimeria tenella (Railliet & Lucet, 1891) Fantham, 1909 from the chicken to other birds have been reviewed by McLoughlin ( 11). Some workers have reported successful cross-transmission without the use of immunosuppressive drugs. These reports, however, are questionable, the results they contain having been attributed to extraneous infections that resulted from poor experimental control. None of the workers looked for tissue stages, and evidence of infection was based only on finding oocysts in the feces. Doran ( 5 ) reported the development of E. tenelta to oocysts in primary kidney cell cultures from chickens, and according to Doran & Augustine ( 6 ) E. tenetlu underwent complete endogenous development in kidney cell cultures from the turkey, ring-necked pheasant, chukar, Japanese quail, and guinea fowl. I n light of the apparent lack of specificity in cell culture, the present study was undertaken to examine more thoroughly host specificity of E. tenella in gallinaceous birds (14).
examined. When oocysts were detected by coverslip flotation, counts were made in McMaster’s chambers. Ten ml of fecal suspension was diluted $6 with 1 M sucrose for a preliminary count. Next, ten 10-ml aliquots of fecal suspension were diluted so that each chamber would contain 100 to 200 oocysts; the principle set forth by Long & Rowell (8) was followed. Two cells/aliquot were counted. If the preliminary count was negative, and this indicated less than 40 oocysts/ml of fecal suspension, the fecal suspension was processed by continuous flow differential density flotation (CFDDF) (13), as were the fecal suspensions examined by coverslip flotation. In all instances, this material was concentrated to 20 ml. Ten ml was examined by the coverslip flotation method and the remaining 10 ml was sporulated and given by gavage to 2 parasite-free chicks. Fecal material from these chicks was collected for 6 days and examined by coverslip flotation for oocysts.
MATERIALS AND METHODS
Only the breeds of G. gullus developed patent infections. No statistical differences were found in the number of oocysts produced/bird among the 6 breeds (Table 2). Subinoculation of fecal material found negative by the coverslip flotation method did not result in oocyst production in susceptible chicks. Tissue sections at 4 hr contained a few sporozoites in the cecal lamina propria of 1 of 3 Beltsville Small White turkeys, 1 of 3 chukars, 1 of 1 peafowl, and 1 of 3 guinea fowl. From one to many sporozoites were detected in sections from all breeds of chicken. The only developmental stages in birds other than the chicken were found in 2 A . graeca at 144 and 168 hr; a few macrogametes were found in sections of the ceca (Figs. 1, 2). T h e developmental stages that would be expected at the various time intervals were observed in tissue sections from all breeds of G. gallus (Fig. 3). Tissue and fecal examinations of all control birds were negative.
Fourtecn breeds of gallinaceous birds representing eight genera (Table 1 ) were reared in isolated brooders until they were 3 weeks old. In the 3 experiments, 3-week-old birds were transferred to other buildings and given 103 ? 4.1 x lo3 sporulated oocysts by gavage. At 4, 24, 48, 72, 96, 120, 144, and 168 hr after inoculation, 1-3 birds of each species, both inoculated and noninoculatcd controls, were killed by cardiac exsanguination. The intestines were removed, and pieces from 5 selected areas were excised and either fixed in Helly’s fixative at 4 C or quenched in 2-methyl butane at -160 C. The 5 selected areas consisted of the duodenum, the jejunum between the bile duct and yolk stalk, the yolk stalk, the ileum, and both the ceca and rectum. Tissues fixed in Helly’s fluid wcrc embedded in paraffin and stained with hematoxylin and eosin, Azure Eosin, Whipf‘s polychrome, and Himes & Moriber’s trichrome. Frozen tissues were sectioned in a cryostat, fixed in acetone (-35 C), air-dried, and examined by the indirect fluorescent antibody technic (2, 7 ) . Fecal material was collected from the surviving birds for the first 6 days of the patent period (120-240 hr postinoculation). Initial examination of fecal material was made by using coverslip flotation of 10 ml of the suspension. If no oocysts were detected, 5 additional coverslips (representing 50 ml of a total fecal suspension of 4 liters) were prepared and
RESULTS
DISCUSSION A working hypothesis for both the cell culture experiments ( 6 ) and the present studies involving avian hosts, suggested by the studies of Lund & Chute ( 9 ) with the histomonadheterakid combination in gallinaceous birds, was that host specificity was relative to genetic proximity : close phylogenetic relatives would be more likely to support a common parasite.
155
HOSTSPECIFICITY OF Eimeria tenella
156
Figs. 13. [Photomicrographs of Eimeria tenella infection in cecum. X 400.1 1. Macrogametes (arrow) in cecal crypt of Alectoris ,qraeca at 144 hr. 2. Macrogamete (arrow) in cecal crypt of Alectoris graeca at 168 hr. 3. One of few recognizable cecal crypts
in White Leghorn, Gallus gallus, at 144 hr. Other parts of section contained many oocysts.
TABLE 1. Taxonomic relationship of galliform birds used.* Family, subfamily, genus, species Superfamily Phasianoidea Family Phasianidae Subfamily Odontophorinae Colinus virginianus (Linnaeus) Subfamily Phasianinae Alectoris graeca (Meisner) Coturnix coturnix Temminek & Schlegel Gallus gallus Linnaeus
Phasianus colchicus Linnaeus Pavo cristatus Linnaeus Family Numididae Numida meleagris Linnaeus Family Meleagrididae Meleagris gallopavo Linnaeus
*From Ref. 12.
Breed
Bobwhite Chukar Japanese Quail Athens Random-bred California White Indian Red Jungle Fowl Light Brahmas New Hampshire White Leghorn Chinese Ring-necked Pheasant Blue Indies Peafowl Pearl Gray Guinea Fowl Beltsville Small White Turkey Wild Turkey
This hypothesis is supported by previous studies involving Eimeria mohauensis Doran & Jahn, 1952 in the genus Dipodomys and other closely or distantly related rodents in the genera Perognathus, Peromyscus, Onychomys, Citellus, Neotoma, Mus, and Rattus ( 4 ) . In this work, Doran showed that E. mohavensis did not infect rodents of genera other than Dipodomys and that species and subspecies varied in susceptibility. This has, more or less, been our standard for host specificity of Eimeria. However, host specificity of avian Eimeria has not been elucidated to the same extent (10, 11).
TABLE 2. Oocyst production in breeds of Gallus gallus for the first 6 days of patent period (120-240 hr).*
Breed Athens Random-bred California White Indian Red Jungle Fowl Light Brahma New Hampshire White Leghorn
* Inoculum
Mean oocysts per bird ( X loa)t
Reproductive Index ( X lo3)
423.33 2 71.07 383.16 k 56.65 372.86 k 44.82 396.55 f 40.17 415.09 f 45.32 421.27 & 48.41
4.11 f 0.69 3.72 k 0.55 3.63 k 0.44 3.85 f 0.39 4.03 f 0.44 4.09 f 0.47
size, 103 & 4.1 x lo3 oocysts. t No significant difference in oocyst production between breeds
(P
> 0.05).
HOSTSPEC:IFICITY OF Bimeria tenetla
Fig. 4. Uistribution map of sympatric part of natural range from Refs. 1, 3 ) .
01
I n one sense, the above hypothesis is supported by the present study-E. tenella did not produce a patent infection in any bird except breeds of G. gallus. Although no variation in susceptibility was found between breeds, the present results correlate with those of Doran ( 4 ) . (Comparison of domestic breeds of chickens with species and subspecies of rodents may not be justifiable.) I n another sense, the hypothesis was not supported. Even though the infection may not be patent, one would expect to find more endogenous stages in close phylogenetic relatives than in more distant relatives. Of the birds examined, the pheasants, Phasianus spp., are considered the closest ancestral relatives of G. gallus (12). No stages, not even sporozoites, however, were detected in pheasants. The only tissue stages other than sporozoites were found in a more distant rrlative, the chukar, Alectoris graeca. Although Alectoris is in the same subfamily as Gallus and Phasianus, it is not considered to be closely related (Table 1 ) ( 1 2 ) . I n his studies with E. mohavensis, Doran ( 4 ) showed that Dipodomys merriami rnerriami Mearns, which is sympatric
AleCtOrlS graeca, ciaiius gaiius
157
and
Phasianus colchtcus
(adapted
with the natural host, Dipodomys panamintinus mohavensis ( Grinnell), although not naturally infected with the coccidium, was twice as susceptible to experimental infection. These 2 species of Dipodomys apparently evolved in the same habitat but developed different habits, and under natural conditions D . m . merriami does not acquire infective oocysts. The natural ranges (presumably where E. tenella evolved in Gallus) of Alectoris, Gallus, and Phasianus are sympatric in part (Fig. 4). Phasianus apparently is not susceptible, and Alectoris is only partly susceptible to infection with E. tenella. Therefore, although Alectoris and Phasianus evolved in the same habitat with similar feeding habits, apparently divergent physiologies developed that led to host specificity. Hence, we can only conclude either that Alectoris is a closer relative of Gallus than hitherto believed or that phylogenetic relationships are less important in host specificity of Eimeria than described earlier in this discussion. Speculations on why E. tenella developed in kidney cell cultures of the various gallinaceous birds and not in vivo in birds
HOSTSPECIFICITY OF Eimeria tenella
158
of the same origin are manifold. Sufficient information, however, is not available to support any of these speculations. The conclusion that E. tenella is as genus-specific as previously demonstrated for rodent Eimeria is justified. ACKNOWLEDGMENT Appreciation for technical assistance is expressed to the late Donald E. Thompson, who prepared the tissues and sections, to Joseph Paige, who participated in this study, and Paul M. Baldwin, who cared for the birds and performed the CFDDF technic. REFERENCES 1. Ali S, Ripley SD. 1968. Handbook of the Birds of India and Pakistan, 10 vols, Oxford University Press, Bombay. 2. Cerna 2. 1967. The dynamics of antibody against Eimeria tenella under the fluorescent microscope. Folia Parasitol., Praha 14, 13-8. 3. Dement’ev GP, Gladkov NA, Isakov IuA, Kartashev NN, Kirikov SV, Mikheev AV, Ptushenko ES. 1952. Birds of the Soviet Union [in Russian], vol. 4. Sovetskaia Nauka, Moskva. 4. Doran DJ. 1953. Coccidiosis in the kangaroo rats of California. Univ. Calif., Berkeley, Publ. Zool. 59, 31-60.
5. ___ 1970. Eimera tenella: From sporozoites to oocysts in cell culture. Proc. Helm. SOL. Wash. 37, 84-92. 6. , Augustine PC. 1973. Comparative development of Eimeria tenella from sporozoites to oocysts in primary kidney cell cultures from gallinaceous birds. 1. ProtomoZ. 20, 658-61. 7. Goldman M. 1968. Fluorescent Antibody Methods. Academic Press. New York. 8. Long PL, Rowell JG. 1958. Counting oocysts of chicken coccidia. Lab. Prac. 7. 515-8. 9. Lund EE. Chute AM. 1974. The reproductive potential of Heterakis gallinarum in various species of galliform biids : Implications for survival of H . gallinarum and Histomonas meleagridis to recent times. Int. J . Parasitol. 4, 455-61. 10. Marquardt WC. 1973. Host and site specificity in the coccidia, in Hammond DM, Long PL, eds., T h e Coccidia, University Park Press, Baltimore, pp. 23-43. 11. McLoughlin DK. 1969. The influence of dexamethosone on attempts to transmit Eimeria meleagrimitis to chickens and E. tenella to turkeys. J . Protorool. 16, 145-8. 12. Peters JL. 1934. Check-List of Birds of the World. Harvard University Press, Cambridge. 13. Vetterling JM. 1969. Continuous-flow differential density flotation of coccidial oocysts and a comparison with other methods. J . Parasitol. 55, 412-7. 14. - 1973. Host specificity studies in eight species of gallinaceous birds. 1. Protorool. 20, 5 10.
J. PROTOZOOL. 2 3 ( 1 ) , 158-164 (1976).
Laboratory and Field Studies on Glugeu stephuni (Hagenmuller), a Microsporidan Parasite of Pleuronectid Flatfishes* ROBERT E. OISON Department of Zoology, Oregon State University, Marine Science Center, Newport, Oregon 97365
SYNOPSIS. The microsporidan Glugea stephani is a common parasite of juvenile English sole (Parophrys uetulus) in Yaquina Bay, Oregon. Field observations indicated that fish became infected only in the upper estuary where summer temperatures were above 15 C and the incidence of infection reached 79.8% in the late fall. Laboratory infections developed and parasite growth occurred only a t or above 15 C. The parasite was successfully transmitted to juvenile English sole by brine shrimp (Artemia salina) and amphipod (Corophium spinicorne) vectors as well as by direct ingestion of spores by the host. Infections that resulted from ingestion of spore-carrying vectors were much heavier than those resulting from the direct ingestion of spores. The speckled sanddab (Citharichthys stigmaeus), a nonpleuronectid flatfish, and chum salmon (Oncorhynchus keta) were refractory to G. stephani infection in the laboratory. Results of this study suggest that G. stephani is potentially lethal to young pleuronectid flatfishes when heavy infections involve the entire intestine and reduce the capacity to absorb nutrients. Under these circumstances, starvation is probably the direct or indirect cause of death. The restriction of infection to fish that reach the upper estuary very likely mitigates the impact of G. stephani caused mortality on the entire English sole population on the Yaquina Bay nursery ground. Index Key Words: Glugea stephani; intestinal parasite of English sole (Parophrys vetulus) ; laboratory transmission; temperature effects.
THE
microsporidan Glugea stephani (Hagenmuller, 1899) Woodcock, 1904, has been reported from juvenile English sole, Parophrys vetulus Girard, in Yaquina Bay, Oregon by Wellings et al. (21) and by Olson & Pratt ( 1 4 ) . I t has also been reported to infect juvenile starry flounder, Platichthys stellatw (Pallas), in northern California (8). Glugea stephani was first recorded from pleuronectid fishes in Europe (5, 9, 22). Infections in winter flouder, Pseudopleuronectes americanus (Walbaum), on the east coast of the United States have been reported by Linton ( 1 1 ) and by Stunkard & Lux (18). Of the microsporidans parasitizing fishes, members of the genus Glugea are among the most intensively studied (1-3,6, This investigation was supported by Research Grant 04-5158-2, from the OSU Sea Grant College Program, NOAA Office of Sea Grant, Dept. of Commerce.
13, 17, 18, 20). This genus is characterized by the formation of 2 spores from a sporont and by the formation of “Glugeacysts” or xenomas, greatly hypertrophied, infected host cells (17). Lom & Weiser ( 1 2 ) have reviewed the genus Glugea and believe it to be a junior synonym of Nosema. Sprague (16) believes the genus Glugea is valid and should be retained. Yaquina Bay has been shown to be an important nursery ground for English sole; large numbers of these fishes occupy the estuary from April through October (14). Initial observations on G. stephani in Yaquina Bay indicated that juvenile English sole in the upper areas of the estuary were more likely to be infected than those in the lower estuary. Further, several parasitized juvenile starry flounders were collected in an emaciated condition that suggested that G. stephani was potentially lethal.
Eimeria tenella: Host Specificity in Gallinaceous Birds J. M. VETTERLING United States Department of Agriculture, Animal Parasitology Institute, Agricultural Research Service, Beltsville, Maryland 20705
SYNOPSIS. Eight species representing 8 genera of gallinaceous birds were used: Alectoris graeca; Colinus virginianus; Coturnix coturnix; Callus gallus; Meleagris gallopavo ; Numidia meleagris; Pavo cristatus; Phasianus colchicus. Threeweek-old birds were dosed with sporulated oocysts of Eimeria tenella Beltsville strain. At 4, 24, 48, 72, 96, 120, 144, and 168 hr after inoculation, 1-3 infected birds and uninoculated controls of each species were killed by cardiac exsanguination. Pieces of intestines were fixed and examined for stages of E. tenella as stained paraffin sections or indirect fluorescent antibody preparations. Oocyst counts were made in droppings collected for the first 6 days of the patent period. Sporozoites were found in the lamina propria of some birds of 5 species at 4 hr postinoculation, but no stages were found thereafter except in the breeds of G . gallus and A . graeca. At 144 and 168 hr postinoculation, a few macrogametes were found in the ceca of 2 A . graeca, but no oocysts were found in the feces. No statistical difference was found between the number of oocysts produced/bird in the breeds of G. gallus examined. It is evident from these observations that E . tenella did not complete its life cycle in several close phylogenetic relatives of G. gallus, even though in other studies this parasite was found to complete its life cycle in cell cultures derived from the same birds. Index Key Words: Eimeria tenella; host specificity; Galliformes; Alectoris graeca; Colinus virginianus; Coturnix coturnix; Gallus gallus; Meleagris gallopavo; Numidia meleagris; Pavo cristatus; Phasianus colchicus.
A
TTEMPTS to transmit Eimeria tenella (Railliet & Lucet, 1891) Fantham, 1909 from the chicken to other birds have been reviewed by McLoughlin ( 11). Some workers have reported successful cross-transmission without the use of immunosuppressive drugs. These reports, however, are questionable, the results they contain having been attributed to extraneous infections that resulted from poor experimental control. None of the workers looked for tissue stages, and evidence of infection was based only on finding oocysts in the feces. Doran ( 5 ) reported the development of E. tenelta to oocysts in primary kidney cell cultures from chickens, and according to Doran & Augustine ( 6 ) E. tenetlu underwent complete endogenous development in kidney cell cultures from the turkey, ring-necked pheasant, chukar, Japanese quail, and guinea fowl. I n light of the apparent lack of specificity in cell culture, the present study was undertaken to examine more thoroughly host specificity of E. tenella in gallinaceous birds (14).
examined. When oocysts were detected by coverslip flotation, counts were made in McMaster’s chambers. Ten ml of fecal suspension was diluted $6 with 1 M sucrose for a preliminary count. Next, ten 10-ml aliquots of fecal suspension were diluted so that each chamber would contain 100 to 200 oocysts; the principle set forth by Long & Rowell (8) was followed. Two cells/aliquot were counted. If the preliminary count was negative, and this indicated less than 40 oocysts/ml of fecal suspension, the fecal suspension was processed by continuous flow differential density flotation (CFDDF) (13), as were the fecal suspensions examined by coverslip flotation. In all instances, this material was concentrated to 20 ml. Ten ml was examined by the coverslip flotation method and the remaining 10 ml was sporulated and given by gavage to 2 parasite-free chicks. Fecal material from these chicks was collected for 6 days and examined by coverslip flotation for oocysts.
MATERIALS AND METHODS
Only the breeds of G. gullus developed patent infections. No statistical differences were found in the number of oocysts produced/bird among the 6 breeds (Table 2). Subinoculation of fecal material found negative by the coverslip flotation method did not result in oocyst production in susceptible chicks. Tissue sections at 4 hr contained a few sporozoites in the cecal lamina propria of 1 of 3 Beltsville Small White turkeys, 1 of 3 chukars, 1 of 1 peafowl, and 1 of 3 guinea fowl. From one to many sporozoites were detected in sections from all breeds of chicken. The only developmental stages in birds other than the chicken were found in 2 A . graeca at 144 and 168 hr; a few macrogametes were found in sections of the ceca (Figs. 1, 2). T h e developmental stages that would be expected at the various time intervals were observed in tissue sections from all breeds of G. gallus (Fig. 3). Tissue and fecal examinations of all control birds were negative.
Fourtecn breeds of gallinaceous birds representing eight genera (Table 1 ) were reared in isolated brooders until they were 3 weeks old. In the 3 experiments, 3-week-old birds were transferred to other buildings and given 103 ? 4.1 x lo3 sporulated oocysts by gavage. At 4, 24, 48, 72, 96, 120, 144, and 168 hr after inoculation, 1-3 birds of each species, both inoculated and noninoculatcd controls, were killed by cardiac exsanguination. The intestines were removed, and pieces from 5 selected areas were excised and either fixed in Helly’s fixative at 4 C or quenched in 2-methyl butane at -160 C. The 5 selected areas consisted of the duodenum, the jejunum between the bile duct and yolk stalk, the yolk stalk, the ileum, and both the ceca and rectum. Tissues fixed in Helly’s fluid wcrc embedded in paraffin and stained with hematoxylin and eosin, Azure Eosin, Whipf‘s polychrome, and Himes & Moriber’s trichrome. Frozen tissues were sectioned in a cryostat, fixed in acetone (-35 C), air-dried, and examined by the indirect fluorescent antibody technic (2, 7 ) . Fecal material was collected from the surviving birds for the first 6 days of the patent period (120-240 hr postinoculation). Initial examination of fecal material was made by using coverslip flotation of 10 ml of the suspension. If no oocysts were detected, 5 additional coverslips (representing 50 ml of a total fecal suspension of 4 liters) were prepared and
RESULTS
DISCUSSION A working hypothesis for both the cell culture experiments ( 6 ) and the present studies involving avian hosts, suggested by the studies of Lund & Chute ( 9 ) with the histomonadheterakid combination in gallinaceous birds, was that host specificity was relative to genetic proximity : close phylogenetic relatives would be more likely to support a common parasite.
155
HOSTSPECIFICITY OF Eimeria tenella
156
Figs. 13. [Photomicrographs of Eimeria tenella infection in cecum. X 400.1 1. Macrogametes (arrow) in cecal crypt of Alectoris ,qraeca at 144 hr. 2. Macrogamete (arrow) in cecal crypt of Alectoris graeca at 168 hr. 3. One of few recognizable cecal crypts
in White Leghorn, Gallus gallus, at 144 hr. Other parts of section contained many oocysts.
TABLE 1. Taxonomic relationship of galliform birds used.* Family, subfamily, genus, species Superfamily Phasianoidea Family Phasianidae Subfamily Odontophorinae Colinus virginianus (Linnaeus) Subfamily Phasianinae Alectoris graeca (Meisner) Coturnix coturnix Temminek & Schlegel Gallus gallus Linnaeus
Phasianus colchicus Linnaeus Pavo cristatus Linnaeus Family Numididae Numida meleagris Linnaeus Family Meleagrididae Meleagris gallopavo Linnaeus
*From Ref. 12.
Breed
Bobwhite Chukar Japanese Quail Athens Random-bred California White Indian Red Jungle Fowl Light Brahmas New Hampshire White Leghorn Chinese Ring-necked Pheasant Blue Indies Peafowl Pearl Gray Guinea Fowl Beltsville Small White Turkey Wild Turkey
This hypothesis is supported by previous studies involving Eimeria mohauensis Doran & Jahn, 1952 in the genus Dipodomys and other closely or distantly related rodents in the genera Perognathus, Peromyscus, Onychomys, Citellus, Neotoma, Mus, and Rattus ( 4 ) . In this work, Doran showed that E. mohavensis did not infect rodents of genera other than Dipodomys and that species and subspecies varied in susceptibility. This has, more or less, been our standard for host specificity of Eimeria. However, host specificity of avian Eimeria has not been elucidated to the same extent (10, 11).
TABLE 2. Oocyst production in breeds of Gallus gallus for the first 6 days of patent period (120-240 hr).*
Breed Athens Random-bred California White Indian Red Jungle Fowl Light Brahma New Hampshire White Leghorn
* Inoculum
Mean oocysts per bird ( X loa)t
Reproductive Index ( X lo3)
423.33 2 71.07 383.16 k 56.65 372.86 k 44.82 396.55 f 40.17 415.09 f 45.32 421.27 & 48.41
4.11 f 0.69 3.72 k 0.55 3.63 k 0.44 3.85 f 0.39 4.03 f 0.44 4.09 f 0.47
size, 103 & 4.1 x lo3 oocysts. t No significant difference in oocyst production between breeds
(P
> 0.05).
HOSTSPEC:IFICITY OF Bimeria tenetla
Fig. 4. Uistribution map of sympatric part of natural range from Refs. 1, 3 ) .
01
I n one sense, the above hypothesis is supported by the present study-E. tenella did not produce a patent infection in any bird except breeds of G. gallus. Although no variation in susceptibility was found between breeds, the present results correlate with those of Doran ( 4 ) . (Comparison of domestic breeds of chickens with species and subspecies of rodents may not be justifiable.) I n another sense, the hypothesis was not supported. Even though the infection may not be patent, one would expect to find more endogenous stages in close phylogenetic relatives than in more distant relatives. Of the birds examined, the pheasants, Phasianus spp., are considered the closest ancestral relatives of G. gallus (12). No stages, not even sporozoites, however, were detected in pheasants. The only tissue stages other than sporozoites were found in a more distant rrlative, the chukar, Alectoris graeca. Although Alectoris is in the same subfamily as Gallus and Phasianus, it is not considered to be closely related (Table 1 ) ( 1 2 ) . I n his studies with E. mohavensis, Doran ( 4 ) showed that Dipodomys merriami rnerriami Mearns, which is sympatric
AleCtOrlS graeca, ciaiius gaiius
157
and
Phasianus colchtcus
(adapted
with the natural host, Dipodomys panamintinus mohavensis ( Grinnell), although not naturally infected with the coccidium, was twice as susceptible to experimental infection. These 2 species of Dipodomys apparently evolved in the same habitat but developed different habits, and under natural conditions D . m . merriami does not acquire infective oocysts. The natural ranges (presumably where E. tenella evolved in Gallus) of Alectoris, Gallus, and Phasianus are sympatric in part (Fig. 4). Phasianus apparently is not susceptible, and Alectoris is only partly susceptible to infection with E. tenella. Therefore, although Alectoris and Phasianus evolved in the same habitat with similar feeding habits, apparently divergent physiologies developed that led to host specificity. Hence, we can only conclude either that Alectoris is a closer relative of Gallus than hitherto believed or that phylogenetic relationships are less important in host specificity of Eimeria than described earlier in this discussion. Speculations on why E. tenella developed in kidney cell cultures of the various gallinaceous birds and not in vivo in birds
HOSTSPECIFICITY OF Eimeria tenella
158
of the same origin are manifold. Sufficient information, however, is not available to support any of these speculations. The conclusion that E. tenella is as genus-specific as previously demonstrated for rodent Eimeria is justified. ACKNOWLEDGMENT Appreciation for technical assistance is expressed to the late Donald E. Thompson, who prepared the tissues and sections, to Joseph Paige, who participated in this study, and Paul M. Baldwin, who cared for the birds and performed the CFDDF technic. REFERENCES 1. Ali S, Ripley SD. 1968. Handbook of the Birds of India and Pakistan, 10 vols, Oxford University Press, Bombay. 2. Cerna 2. 1967. The dynamics of antibody against Eimeria tenella under the fluorescent microscope. Folia Parasitol., Praha 14, 13-8. 3. Dement’ev GP, Gladkov NA, Isakov IuA, Kartashev NN, Kirikov SV, Mikheev AV, Ptushenko ES. 1952. Birds of the Soviet Union [in Russian], vol. 4. Sovetskaia Nauka, Moskva. 4. Doran DJ. 1953. Coccidiosis in the kangaroo rats of California. Univ. Calif., Berkeley, Publ. Zool. 59, 31-60.
5. ___ 1970. Eimera tenella: From sporozoites to oocysts in cell culture. Proc. Helm. SOL. Wash. 37, 84-92. 6. , Augustine PC. 1973. Comparative development of Eimeria tenella from sporozoites to oocysts in primary kidney cell cultures from gallinaceous birds. 1. ProtomoZ. 20, 658-61. 7. Goldman M. 1968. Fluorescent Antibody Methods. Academic Press. New York. 8. Long PL, Rowell JG. 1958. Counting oocysts of chicken coccidia. Lab. Prac. 7. 515-8. 9. Lund EE. Chute AM. 1974. The reproductive potential of Heterakis gallinarum in various species of galliform biids : Implications for survival of H . gallinarum and Histomonas meleagridis to recent times. Int. J . Parasitol. 4, 455-61. 10. Marquardt WC. 1973. Host and site specificity in the coccidia, in Hammond DM, Long PL, eds., T h e Coccidia, University Park Press, Baltimore, pp. 23-43. 11. McLoughlin DK. 1969. The influence of dexamethosone on attempts to transmit Eimeria meleagrimitis to chickens and E. tenella to turkeys. J . Protorool. 16, 145-8. 12. Peters JL. 1934. Check-List of Birds of the World. Harvard University Press, Cambridge. 13. Vetterling JM. 1969. Continuous-flow differential density flotation of coccidial oocysts and a comparison with other methods. J . Parasitol. 55, 412-7. 14. - 1973. Host specificity studies in eight species of gallinaceous birds. 1. Protorool. 20, 5 10.
J. PROTOZOOL. 2 3 ( 1 ) , 158-164 (1976).
Laboratory and Field Studies on Glugeu stephuni (Hagenmuller), a Microsporidan Parasite of Pleuronectid Flatfishes* ROBERT E. OISON Department of Zoology, Oregon State University, Marine Science Center, Newport, Oregon 97365
SYNOPSIS. The microsporidan Glugea stephani is a common parasite of juvenile English sole (Parophrys uetulus) in Yaquina Bay, Oregon. Field observations indicated that fish became infected only in the upper estuary where summer temperatures were above 15 C and the incidence of infection reached 79.8% in the late fall. Laboratory infections developed and parasite growth occurred only a t or above 15 C. The parasite was successfully transmitted to juvenile English sole by brine shrimp (Artemia salina) and amphipod (Corophium spinicorne) vectors as well as by direct ingestion of spores by the host. Infections that resulted from ingestion of spore-carrying vectors were much heavier than those resulting from the direct ingestion of spores. The speckled sanddab (Citharichthys stigmaeus), a nonpleuronectid flatfish, and chum salmon (Oncorhynchus keta) were refractory to G. stephani infection in the laboratory. Results of this study suggest that G. stephani is potentially lethal to young pleuronectid flatfishes when heavy infections involve the entire intestine and reduce the capacity to absorb nutrients. Under these circumstances, starvation is probably the direct or indirect cause of death. The restriction of infection to fish that reach the upper estuary very likely mitigates the impact of G. stephani caused mortality on the entire English sole population on the Yaquina Bay nursery ground. Index Key Words: Glugea stephani; intestinal parasite of English sole (Parophrys vetulus) ; laboratory transmission; temperature effects.
THE
microsporidan Glugea stephani (Hagenmuller, 1899) Woodcock, 1904, has been reported from juvenile English sole, Parophrys vetulus Girard, in Yaquina Bay, Oregon by Wellings et al. (21) and by Olson & Pratt ( 1 4 ) . I t has also been reported to infect juvenile starry flounder, Platichthys stellatw (Pallas), in northern California (8). Glugea stephani was first recorded from pleuronectid fishes in Europe (5, 9, 22). Infections in winter flouder, Pseudopleuronectes americanus (Walbaum), on the east coast of the United States have been reported by Linton ( 1 1 ) and by Stunkard & Lux (18). Of the microsporidans parasitizing fishes, members of the genus Glugea are among the most intensively studied (1-3,6, This investigation was supported by Research Grant 04-5158-2, from the OSU Sea Grant College Program, NOAA Office of Sea Grant, Dept. of Commerce.
13, 17, 18, 20). This genus is characterized by the formation of 2 spores from a sporont and by the formation of “Glugeacysts” or xenomas, greatly hypertrophied, infected host cells (17). Lom & Weiser ( 1 2 ) have reviewed the genus Glugea and believe it to be a junior synonym of Nosema. Sprague (16) believes the genus Glugea is valid and should be retained. Yaquina Bay has been shown to be an important nursery ground for English sole; large numbers of these fishes occupy the estuary from April through October (14). Initial observations on G. stephani in Yaquina Bay indicated that juvenile English sole in the upper areas of the estuary were more likely to be infected than those in the lower estuary. Further, several parasitized juvenile starry flounders were collected in an emaciated condition that suggested that G. stephani was potentially lethal.
