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Development of Caryospora bigenetica (Apicomplexa:Eimeriorina) in experimentally infected mice
DE~LOPMENT OF CA~Y~~~~~ ~~G~~~~~CA (APICOMPLEXA:EIMERIORINA) IN EXPERIMENTALLY INFECTED MICE STEVEJ. UPTON* and SUSAN M. BARNARD? * Division
of Biology, Ackert Hall, Kansas State University, Manhattan, KS 66506, U.S.A.
t Zoo Atlanta, Atlanta, GA 30315, U.S.A. (Received 11 February 1987)
Abstract--C’PToN S. J. and BARNARD S. M. 1988. Development
of Curyospora bjge~et~c~ (Apicompl~x~:Eimeriorina~ in expe~mentally infected mice. ~nternationff~ Journal for Purus~folo~ 18: 15-20. Developmental stages of Curyospura bigenetica were found in the connective tissues of the cheek, nose, tongue eastern
and scrotum of laboratory mice experimentally infected per OS with oocysts derived from the diamondback rattlesnake, Crotalus adamanteus, or northern copperhead, Agkistrodon contortrix mokusen. Type I meronts were found at 8 days post-inoculation (DPI), whereas type 11and type III meronts were seen at 10 DPI. Microgametocytes and macrogametes were observed at 10 and 12 DPI, unsporulated oocysts at 1 O-1 4 DPI, and sporulated oocysts at I2 and 14 DPI. Caryocysts (monozoic cysts) were first found 12 DPI and persisted throughout the duration of the study. Four mice, each injected intraperitoneally with 4.0 X 10” free sporozoites and examined at 8 and 10 DPI, also had developmental stages of C. bigenetica in the connective tissue of the cheek, nose and tongue. INDEX KEY WORDS: Caryospora bigeneticu; coccidia; teus; Agktitrodon contortrix; Serpentes; Crotalidae; mice.
INTRODIJCTION
bigenetica (Apicomplexa: Eimeriorina) was described originally by Wacha & Christiansen (1982) from the timber rattlesnake, Crotufus horridus, and Massasauga, S~~trurus e~te~~atl~, from Iowa. When sporulated oocysts were fed to laboratory white mice, Mus musculus, gamogony and sporogony were found on days 8 and 10 post-inoculation, respectively, in the connective tissues of the cheek and tongue. Sporozoites resulting from sporogony were found to become located in host cells as monozoic cysts, termed ‘caryocysts’. No evidence of merogony was found in infected mice. Upton, Current, Ernst & Barnard (1984) experimentally infected laboratory mice with Caryosporu simplex, a morphologically similar coccidian occurring in viperids. Development of the parasite was similar to that reported by Wacha & Christiansen (1982) for C: bigenetica, however, two generations of merogony were noted prior to gamogony and sporogony. Since 1982, we have examined feces from a variety of crotalids collected from various regions of the United States. Many of these snakes, including the western Massasauga (Sistrurus catenutus tergeminus), eastern diamondback rattlesnake (Crotalus adumunteus), southern copperhead (Agkistrodon contortrix contortrix), and the northern copperhead (A. c. mokasen), have been found to be passing oocysts Culyosporu
Apicomplexa;
Eimeriidae;
Crotnlus rtdumcm-
morphologically indistinguishable from Caryospora bigenetica Wacha & Christiansen, 1982. In this paper, oocysts derived from the feces of naturally infected C. adamanteus and A. c. mokasen, snakes collected in Georgia and New Jersey, U.S.A., respectively were used to infect laboratory mice and to support and supplement previous work by Wacha & Christiansen (1982) on the life cycle of C. bigeneticu.
MATERIALS
AND METHODS
Preparation of oocysts, sporocysts ctnd sporozoitesFeces from a naturally infected eastern diamondback rattlesnake, C. adumnnteus, and from a naturally infected northern copperhead, A. c. mokusen, both of which contained numerous unsporulated oocysts of C. bigeneticu, were placed in individual Petri dishes in a thin layer of 2.5% (w/v) aqueous potassium dichromate solution (KICrlO,) for 1 week at 27-30°C. Sporulated oocysts were removed from fecal debris by straining the feces through a graded series of wire mesh sieves, counted with a haemacytometer, and stored in 2.5% (w/v) aqueous K2Cr20, solution at 4°C for 2 months. Prior to use, oocysts were washed 3 times in tap water by centrifugation. Free sporocysts were obtained by resuspending some of the ovcysts obtained from C. adumantetts in phosphate buffered saline (PBS) (pH 7.2) and removing the oocyst walls in a motor-driven Teflon-coated tissue grinder for 20 s. Sporozoites were obtained by incubating free sporocysts in a 0.25% (w/v) trypsin-0.75% (w/v) sodium taurocholate/PBS solution for 30 min. followed by column filtration (Tilahun & Stockdale. 1982).
16
S. 3. UPTON and S. M. BARNARD
experimental animals. All mice used for experimental inocufations with oocysts, sporocysts, or sporozoites from C. adamanteus were male, CD-l, M. musculus, and were 68 weeks old at the time of the experiment. Mice inoculated with oocysts from A. c mokasen were male, Swiss-Webster, A4. musculus, also 6-8 weeks of age. All mice were housed in groups of two to four in metal cages on a 12 h light/dark cycIe and were supplied with sterile wood shavings for bedding and given commercial rodent pellets and water ad l~bi~arn Animal inoculafjo~s. Twenty-seven CD- 1 mice were each inoculated by stomach tube with 3.0 X 10” sporulated oocysts of C. bigenefica obtained from the feces of C. adamanteus. At 2, 4, 6, 8, IO, 12, 14, 16 and 20 days postinoculation (DPI), three of the mice were killed by cervical dislocation. Six additional mice were each inoculated per OS with a mixture of 3.1 X 10’ oocysts and 1.l X IO5 sporocysts of the same isolate. Two mice from this group were killed as above at 6,8 and 10 DPI. Four additional mice were also infected by injecting each mouse intra-peritoneaily (IP) with 4.0 X 105 sporozoites of the same isolate. Two mice from this group were killed by cervical dislocation at 8 and 10 DPI. Six uninoculated, CD-l mice served as controls and pairs of mice were killed as above at 8, 12 and 16 DPI. Portions of dermal connective tissues from the cheeks, tongue, nose, scrotum and dorsum were placed in AFA [alcohol (3 parts of 95% ethanol)-formalin (1 part stock solution)-glacial acetic acid (1 part)-distilled water (5 parts)] fixative and processed for routine histological sectioning and staining with hematox~lin and eosin. Squash preparations were made of additional portions of the-same tissues at 10 and 12 DPI and were examined bv brirhtfield microscoov for developmental stages of the p&as&. Measurements-of 25 parasites were made for each developmental stage noted in sectioned material. All measurements were made using a calibrated ocular micrometer and are expressed as means followed by the range in parentheses. Ten Swiss-Webster mice were inoculated per OS with 2.0 X 10 sporulated oocysts of C: h&nerica, however. this isolate was derived from the feces of A. c. mokasen. Pairs of mice were killed by cervical dislocation at 10, 12. 14. 40 and 60 DPI. Squash preparations wcrc made from portions of the cheek, tongue and nose connective tissue from each mouse and examined
for live parasitic stages by light microscopy interference contract (NIC) illumination.
using Nomarski
RESULTS
of
Parasites were not found in histological sections or squash preparations prior to 8 DPI. However, from 8 to 60 DPI, developmental stages of C. bigetzetku were found in fibroblast-like cells of the connective tissues of the cheek. nose, scrotum and less frequently the tongue of all inoculated mice. Mice inoculated orally with a mixture of oocysts and sporocysts appeared subjectively to have larger numbers of parasites than did mice inoculated only with oocysts. Mice inoculated IP with sporozoites alone appeared to have only a moderate number of parasites in the connective tissues. Infected tissues were noticeably swotien at 8-l 6 DPI and were heavily i~ltrated with in~ammatory cells. ~xtraint~tina~ stages. The dimensions and kinetics of each developmental stage of C. bigenetica detected in the histological sections are summarized in Table 1. Qpe I meronts and merozoites. Type I meronts were observed in histological sections at 8 DPI (Fig. 1) and were infrequently encountered. The meronts were spherical or ovoid in shape and contained X- 12 (modes equally of 8 and 10) merozoites. An indistinct, spherical or ovoid ~ytopIasmic mass was usually present with dimensions of 2.8 X 2.7 ‘urn (2.0-4.0 X 2.0-3.0 pm), from which the merozoites appeared to arise by ectomerogony. Usually one (but occasionally two) meront was present within the same host cell. Each meront was located within a spherical or ovoid parasitophorous vacuole. Type I merozoites were elongate, lying parallel within the parasitophorous vacuole and contained a centrally located nucleus and no refractile bodies. 7&e II rneronts atld merozoites. Type I1 meronts were observed in histological sections at if) DPI (Fig.
Sites
infection.
TABLE I--UIMENSIOF;S (IF. FITI) ANIITIMES OP APPEARANCEUC DEVELOPMENTALSTAGI,S OF
C’qosporu bigenerica~u EXPERIMENTALLY INFECTEDCD-l ~tct
Developmental
stage
Type I meronts Type I merozoites Type II meronts Tvpe II merozoites Type III meronts Type 111merozoites Microgametocytes Macrogametes ~n~poruIated oocysts Sporulated oocysts Sporocysts Sporozoites Residuum Spororoites in caryocvsts
Days post-inocuiation
8 10 I0 IU 10 IO, 12
IO.12 IO-14 12.14
12-I-I
Dimension (length~width) (n = 25) 7.5 (iO.O-6.0) x 4.7 (3.5-5.0) X 12.6 (9.0-17.0) x 4.3 (4.0-5.0) x 9.5 (7.0-10.5) x .i.h (3.0-4.2) X 6.2 (4.0~8.0) x 9.5 (7.0-I I .O) x 10.9 (9.G IX) 8.4 (7.5595) 7.1 (6.0-9.0)
6.7 0.9 9.8 1 .o 8.3 1.0 5.x
9.3
(5.0-8.0) (0.X-1.2) (7.0-12.0) (0.X-1.2) (6.0-10.0) (0.X-1.2) (4.0-7.0) (7.0-l 1.0)
x IO.6 (W-l 2.0) x x
7.6 (6.0-X.0) 1.5 (1.2-2.1))
6.5 (5.0-8.0) x 4.1 (3.0-5.0) 5.9 (5.0-7.0) x I .o (I .s-7.0)
Development of C. bigenetica
2) and were more numerous than type I meronts. The type II meronts were spherical or ovoid in shape, larger in size than type I meronts and contained 22-28 (mode = 24) merozoites. The meronts usually contained an indistinct, spherical or ovoid cytoplasmic mass with dimensions of 3.4 X 3.0pm (2.0-6.0 X 2.0-5.0 pm) from which the merozoites appeared to arise by ectomerogony. Usually one (rarely two) type II meronts were present within the same host cell within a spherical or ovoid parasitophorous vacuole. Type II merozoites were moderately elongate and contained a centrally located nucleus and no refractile bodies. Type III meronts and merozoites. Type III meronts were observed in histological sections at 10 DPI (Fig. 3) and were by far the most numerous of all asexual stages seen. They were therefore regarded as the oldest or third asexual generation. These meronts were spherical or ovoid and contained 12-18 (mode = 16) merozoites. An indistinct, spherical or ovoid cytoplasmic mass was usually present measuring 2.0 X 1.9pm (1.0-4.0 X l.O-3.0pm) in size from which the merozoites appeared to arise by ectomerogony. Usually one (but up to three) meronts were found within the same host cell and each was located within a spherical or ovoid parasitophorous vacuole. Type III merozoites were short and stout and contained a centro-posteriorly located nucleus and no refractile bodies. Microgametocytes. Microgametocytes were found as early as 10 DPI, but were most numerous at 12 DPI (Fig. 4). They occurred singly or in groups of up to three and were frequently encountered in areas of connective tissue that contained macrogametes. Microgametocytes were spherical or ovoid in shape and contained 8-14 (mode = 10) microgametes surrounding a spherical or ovoid residuum and lay within a distinct parasitophorous vacuole. Macrogametes. Macrogametes were found as early as 10 DPI. but were most frequently encountered at 12 DPI. The cytoplasm of the macrogamont was lightly granular (Fig. 5) although the size and granularity of these stages was observed to increase with maturity. When fully mature, macrogametes were spherical or ovoid in shape and contained numerous small, cytoplasmic granules, a large prominent nucleus and were located within a distinct parasitophorous vacuole (Fig. 6). Oocysts. Unsporulated oocysts were observed at lo-14 DPI (Fig. 7). The oocyst had a granular cytoplasm and contained one to many homogenous eosinophilic globules. As development proceeded, the sporont contracted and eight sporozoites were eventually formed within the oocyst (Figs. 8 and 9). Sporogony occurred within the sporont membrane and without the formation of a true sporocyst wall, Stieda or substieda bodies. Fully sporulated oocysts were observed at 12 and 14 DPI and had a thin oocyst wall that was observed only in squash preparations. Sporozoites lay parallel to one another and were
17
partially twisted in a semi-spiral around an ovoid, granular residuum. The sporozoites each had a centrally located nucleus and two refractile eosinophilic bodies, each located at either side of the nucleus. Caryocysts. Free sporozoites and caryocysts were first seen at 12 DPI and the caryocysts persisted throughout the duration of the study. Initially, sporozoites separated from the sporocyst residuum, became scattered within the oocyst and finally crossed through the thin oocyst wall (Fig. 10). Sporozoites in caryocysts (Figs. 11 and 12) were within a parasitophorous vacuole and were usually arranged parallel to the long axis of the host cell. Host cells measured 11.1 X 9.9 pm (9.0-14.0 X 8.0-13.0 pm) in size and contained a crescent-horseshoe shaped nucleus usually indicative of macrophages. Caryocysts normally contained a single sporozoite, however, up to four sporozoites were sometimes observed within the same infected host cell. The margin of the host cell usually appeared thicker and darker than the enclosed cytoplasm. Infections with oocysts derivedfrom A. c. mokasen. Examination of mouse tissues at 10,12,14,40 and 60 DPI with oocysts from A. c. mokasen (Figs. 13 and 14) revealed developmental stages of C. bigenetica similar to those found in mice infected with isolates from C. adamanteus. The oocysts were thin walled (Fig. 15) and the sporozoites in fully sporulated oocysts surrounded an ovoid residuum but a true sporocyst wall, Stieda and substieda bodies were not present (Fig. 16). By 40 DPI, however, one of the refractile bodies had disappeared (Figs. 17 and 18) while the larger of the two refractile bodies remained intact. DISCUSSION
Results of our experimental inoculations of mice with oocysts of C. bigenetica are similar, but not identical, to results obtained by Wacha & Christiansen (1982). The present study reports, for the first time, three generations of merogony prior to gamogony in mice inoculated with oocysts. The failure of Wacha & Christiansen (1982) to find asexual stages may best be explained by the fact that they only used 6.0 X 10’ oocysts as inoculating doses whereas we used at least 50 times this number. In addition, we found the times of appearance of gametes and sporulated oocysts to be delayed by approx. 48 h than reported previously. This difference may be explained by the differences in host strain, strain of parasite, numbers of parasites used for experimental inoculations, environmental conditions under which the experiments were conducted or other undetermined factors. However, we are confident that the isolates used in the present study were C. bigenetica. Not only were the isolates found in crotalids, but the oocysts, sporocysts and sporozoites are morphologically indistinguishable from those reported by Wacha & Christiansen (1982).
S. J. UPTON and S. M. BARNARD
18
FIGis. 1-12.
Brightfield photomicrographs experimentally infected FE.
of developmental stages of Caryospora bigeneticrr in the cheek CD-I mice. I-Iematoxylin and eosin stain. (Scale bar = 5 pm.)
I. Type I meront, 8 DPI. Notemerozoites FIG. 2. Type II meront,
10 DPI.
Pm. 3. Type III meront.
10 DPI.
Fro. 4. Micr~~gamet(~cyte,
12 DPI.
Fro. 6. Macrogamete,
12 DPI.
FIG. 7. LJnsporulated FICX. 8 and 9. Sporulated
oocysts,
12 DPI. Note refractile
FIG. 10. Sporulated PK. 1 I. Caryocyst FIG.
12. Caryocyst
(mz).
12 DPI. Note microgametes
Fro. 5. Macrogamont,
oocyst,
(ct), with longitudinalview (ct), with end-on-view
(mi).
12 DPI.
bodies (rb), sporozoites
oocyst with dispersed
dermis
sporozoites ofsporozoite of sporozoite
(sp), and sporocyst
(sp), 14 DPI. (sp), 16 DPI. (sp),
i 6 DPI.
residuum
(sr).
of
Development
of C. bigenetica
19
FIGS. 13-18. Nomarski interference contrast photomicrographs of Catyospora bigeneticu. FIGS. 13 and 14. Oocysts derived from the feces of Agkisfrodon contortrixmokasen. FIGS. 15-l 8. Developmental stages in cheek connective tissues of experimentally infected Swiss-Webster mice. (Scale bar = 5~ m.) FIG. 13. Sporulated FIG. 14. Sporulated FIG. 15. Unsporulated
oocyst, longitudinal
oocyst with refractile
FIG. 16. Sporulated Frc. 17 and 18. Caryocysts
view. Note oocyst wall (ow).
oocyst, end-on-view.Note
oocyst with granular with sporozoites
polar granule(pg).
globule (rg), large nucleus (n), and thin oocyst wall (ow), 10 DPI sporocyst
residuum
(sr) and sporozoites
(sp), each with a nucleus (n) and a refractile
The two species, C. bigenetica and C. simplex, appear to be closely related on the basis of their morphological and developmental characteristics. However, we have noted several differences which could be used to distinguish between the two species. Oocysts of C. bigenetica derived from snakes appear to have a slightly thinner wall than oocysts of C. simplex. The Stieda body of C. bigenetica also appears to be slightly smaller than that of C. simplex and, more distinctively, the positions of the refractile bodies in each species are different; those of C. bigenetica being anterior and central while in C. simplex they are central and posterior (cf. Upton, Current & Barnard, 1986). Pathologically, we have noted that the cheeks of mice appear to be more heavily infiltrated with inflammatory cells and are considerably more swollen during C. bigeneticu infections, even when infections are compared using
(sp), 14 DPI. body (rb), 40 DPI.
low doses (5.0 X 10J oocysts) of C. bigenetica vs. big: doses (2.5 X 105) of C. simplex (unpublished data). Finally, Upton and Barnard (1986) have shown that oocysts of C. simplex readily infect Palestine vipers, Viper-a xanthina palestinae. Although attempts to infect North American crotalids with C. simplex were not completed nor presented in the report, we have since found that eastern diamondback rattlesnakes, C. adamanteus, fail to become infected with oocysts of C. simplex (unpublished data). Thus, C. bigenetica and C. simplex appear to represent two distinct species. Wacha & Christiansen (1982) reported two types of meronts in snakes. Type I meronts which contained 12 (10-14) merozoites and a residuum and type II meronts containing seven (four to nine) merozoites that lay either parallel or in a staggered formation within the parasitophorous vacuole with no observ-
20
S. J. UPTON and S. M. BAKNARD
able residuum. In the present study, three types of meronts were seen. At 8 DPI, type I meronts were present and contained 8-12 slender, parallel merozoites. At IO DPI, large type II meronts, with about 22-28 merozoites and the highly numerous type II1 meronts, with about 12-18 merozoites were seen. Comparing data from the two studies, we are unable to ascertain whether the two types of meronts found in the intestine of snakes correspond to any of the meronts we observed. It is possible that no retationship exists between the different generations of meronts that occur in the different hosts. By 8 DPI, we noted that all mice inoculated with oocysts appeared lethargic and their facial tissues, tongue and scrotum appeared edematous. Wacha & Christiansen (1982) noted a similar condition in the facial tissues of mice they infected. However, two of the five mice they inoculated died on about 10 DPI whereas we have never observed any mortality due to C. bigeneticu in over 100 mice inoculated with various doses over the last 2 years, including CD- 1, BALB/c, CS7BL/6, and Swiss-Webster mice. Since our previous work (Upton et al., 1984), we have inoculated additional mice with C. simplex and have found that this coccidium also develops in the scrotal dermis of mice. The finding that both C. simplex and C. bige~etjca develop in facial, tongue, and scrotal connective tissues suggests the selection of these predilection sites may be temperaturedependent. These areas tend to be slightly cooler regions of the body. Although infections are observably uncomfortable for mice, the location of parasites in these sites may allow for ready access to intestinal cells in the snake during the early stages of digestion. Marcone (1908) reported subcut~eous nodular coccidiosis from a dog from Italy and he named this coccidian “Coccidium nudum”. Shelton, Kinter & MacKintosh (1968) and Sangster, Styler & Hall (1985) also reported similar lesions from dogs in Missouri and Georgia, U.S.A., respectively. All three of these reports not only describe dermal meronts, but Eimeriidae/Sarcocystidae-like macrogametes and microgametocytes as well. It is interesting to note that
the dog studied by Shelton et al. (1968) died 2 months later of acute canine distemper, a virus reputed to cause immunodepression in canids. The fact that C. simplex and C. bigenetica both develop in the intestinal tract of poikilotherms (snakes) and, experimentally, in the connective tissues of homeotherms (rodents) suggests that, under the appropriate conditions, both species (and perhaps other Caryospwu spp. as well) may have the potential to become nonhost specific in immunosuppressed hosts. if this is the case, it is possible that the Curyuspora may represent the causative agents of canine subcutaneous nodular coccidiosis.
MARCONE G.
REFERENCES 1908. Sporozoen-Dermatosen des Hundes.
~eitschri~ fiir Infektionskrurzkheiten, Purus~te~krz~nkheiten z&d ~~ie~~ der Haustiere 4: 5-32. SANGSTER
Coccidia
L.T..
STYLER E. L. oi HALL G. A. 19%. associated with cutaneous nodules in a dog.
Veterinary Pathology 22: SHELTON
186-188.
G. C., KINTER L. D. & MACKINTOSH D. 0. 1968.
A coccidia-like organism associated with subcutaneous granulomata in dog. Journul of’the American Veterinag
MediculAssociation TIUHUN
152: 263-267.
G. & STOCKDALE P. I-1. G. 1982. Sensitivity
and specificity of the indirect fluorescent antibody test rn the study of four murine coccidia. Joz~r~a~of ~r~f~~aoZ~,~~
29: 129-132. ~JPTON S. J., CURRENT W. L., J.5~~ls-rJ. V. &BARNARD S. M.
1984. Extraintestinal development of C’uryosporu sbnpke.w (Apicomplexa:Eimeriidae) in experimentally infected mice, Mus musczdzrs. Joztrrzal of t’rotozoolog_v3 1: 3X398. UPTOK S. J. & BARNARD S. M. 1986. Experimental transof simplex mission Cff ~osporcr (Apicomplexa: Eimeriidae) to Palestine vipers, t’$rt-44 ,~atzt~zitz~z palestinae (Serpentes: Viperidae). Jozrrnai of Proio-
;oologv 33: 129-130. UPTON S. J., CURRENT W. L. s( BARNARD S. M. 19X6. A review of the genus Curyospora (Apicomplexa:limeriidae). Systematic Parasitology 8: 3-2 1. Wacw, R. S. & CHRISTIANSEN J. L. 19X2. Development of Chtyospom bigeneticu n. sp. (Apicomplexa:Eimeriidae) in rattlesnakes and laboratory mice. Jownal of‘ frotozoolop 29: 272-278.
of Biology, Ackert Hall, Kansas State University, Manhattan, KS 66506, U.S.A.
t Zoo Atlanta, Atlanta, GA 30315, U.S.A. (Received 11 February 1987)
Abstract--C’PToN S. J. and BARNARD S. M. 1988. Development
of Curyospora bjge~et~c~ (Apicompl~x~:Eimeriorina~ in expe~mentally infected mice. ~nternationff~ Journal for Purus~folo~ 18: 15-20. Developmental stages of Curyospura bigenetica were found in the connective tissues of the cheek, nose, tongue eastern
and scrotum of laboratory mice experimentally infected per OS with oocysts derived from the diamondback rattlesnake, Crotalus adamanteus, or northern copperhead, Agkistrodon contortrix mokusen. Type I meronts were found at 8 days post-inoculation (DPI), whereas type 11and type III meronts were seen at 10 DPI. Microgametocytes and macrogametes were observed at 10 and 12 DPI, unsporulated oocysts at 1 O-1 4 DPI, and sporulated oocysts at I2 and 14 DPI. Caryocysts (monozoic cysts) were first found 12 DPI and persisted throughout the duration of the study. Four mice, each injected intraperitoneally with 4.0 X 10” free sporozoites and examined at 8 and 10 DPI, also had developmental stages of C. bigenetica in the connective tissue of the cheek, nose and tongue. INDEX KEY WORDS: Caryospora bigeneticu; coccidia; teus; Agktitrodon contortrix; Serpentes; Crotalidae; mice.
INTRODIJCTION
bigenetica (Apicomplexa: Eimeriorina) was described originally by Wacha & Christiansen (1982) from the timber rattlesnake, Crotufus horridus, and Massasauga, S~~trurus e~te~~atl~, from Iowa. When sporulated oocysts were fed to laboratory white mice, Mus musculus, gamogony and sporogony were found on days 8 and 10 post-inoculation, respectively, in the connective tissues of the cheek and tongue. Sporozoites resulting from sporogony were found to become located in host cells as monozoic cysts, termed ‘caryocysts’. No evidence of merogony was found in infected mice. Upton, Current, Ernst & Barnard (1984) experimentally infected laboratory mice with Caryosporu simplex, a morphologically similar coccidian occurring in viperids. Development of the parasite was similar to that reported by Wacha & Christiansen (1982) for C: bigenetica, however, two generations of merogony were noted prior to gamogony and sporogony. Since 1982, we have examined feces from a variety of crotalids collected from various regions of the United States. Many of these snakes, including the western Massasauga (Sistrurus catenutus tergeminus), eastern diamondback rattlesnake (Crotalus adumunteus), southern copperhead (Agkistrodon contortrix contortrix), and the northern copperhead (A. c. mokasen), have been found to be passing oocysts Culyosporu
Apicomplexa;
Eimeriidae;
Crotnlus rtdumcm-
morphologically indistinguishable from Caryospora bigenetica Wacha & Christiansen, 1982. In this paper, oocysts derived from the feces of naturally infected C. adamanteus and A. c. mokasen, snakes collected in Georgia and New Jersey, U.S.A., respectively were used to infect laboratory mice and to support and supplement previous work by Wacha & Christiansen (1982) on the life cycle of C. bigeneticu.
MATERIALS
AND METHODS
Preparation of oocysts, sporocysts ctnd sporozoitesFeces from a naturally infected eastern diamondback rattlesnake, C. adumnnteus, and from a naturally infected northern copperhead, A. c. mokusen, both of which contained numerous unsporulated oocysts of C. bigeneticu, were placed in individual Petri dishes in a thin layer of 2.5% (w/v) aqueous potassium dichromate solution (KICrlO,) for 1 week at 27-30°C. Sporulated oocysts were removed from fecal debris by straining the feces through a graded series of wire mesh sieves, counted with a haemacytometer, and stored in 2.5% (w/v) aqueous K2Cr20, solution at 4°C for 2 months. Prior to use, oocysts were washed 3 times in tap water by centrifugation. Free sporocysts were obtained by resuspending some of the ovcysts obtained from C. adumantetts in phosphate buffered saline (PBS) (pH 7.2) and removing the oocyst walls in a motor-driven Teflon-coated tissue grinder for 20 s. Sporozoites were obtained by incubating free sporocysts in a 0.25% (w/v) trypsin-0.75% (w/v) sodium taurocholate/PBS solution for 30 min. followed by column filtration (Tilahun & Stockdale. 1982).
16
S. 3. UPTON and S. M. BARNARD
experimental animals. All mice used for experimental inocufations with oocysts, sporocysts, or sporozoites from C. adamanteus were male, CD-l, M. musculus, and were 68 weeks old at the time of the experiment. Mice inoculated with oocysts from A. c mokasen were male, Swiss-Webster, A4. musculus, also 6-8 weeks of age. All mice were housed in groups of two to four in metal cages on a 12 h light/dark cycIe and were supplied with sterile wood shavings for bedding and given commercial rodent pellets and water ad l~bi~arn Animal inoculafjo~s. Twenty-seven CD- 1 mice were each inoculated by stomach tube with 3.0 X 10” sporulated oocysts of C. bigenefica obtained from the feces of C. adamanteus. At 2, 4, 6, 8, IO, 12, 14, 16 and 20 days postinoculation (DPI), three of the mice were killed by cervical dislocation. Six additional mice were each inoculated per OS with a mixture of 3.1 X 10’ oocysts and 1.l X IO5 sporocysts of the same isolate. Two mice from this group were killed as above at 6,8 and 10 DPI. Four additional mice were also infected by injecting each mouse intra-peritoneaily (IP) with 4.0 X 105 sporozoites of the same isolate. Two mice from this group were killed by cervical dislocation at 8 and 10 DPI. Six uninoculated, CD-l mice served as controls and pairs of mice were killed as above at 8, 12 and 16 DPI. Portions of dermal connective tissues from the cheeks, tongue, nose, scrotum and dorsum were placed in AFA [alcohol (3 parts of 95% ethanol)-formalin (1 part stock solution)-glacial acetic acid (1 part)-distilled water (5 parts)] fixative and processed for routine histological sectioning and staining with hematox~lin and eosin. Squash preparations were made of additional portions of the-same tissues at 10 and 12 DPI and were examined bv brirhtfield microscoov for developmental stages of the p&as&. Measurements-of 25 parasites were made for each developmental stage noted in sectioned material. All measurements were made using a calibrated ocular micrometer and are expressed as means followed by the range in parentheses. Ten Swiss-Webster mice were inoculated per OS with 2.0 X 10 sporulated oocysts of C: h&nerica, however. this isolate was derived from the feces of A. c. mokasen. Pairs of mice were killed by cervical dislocation at 10, 12. 14. 40 and 60 DPI. Squash preparations wcrc made from portions of the cheek, tongue and nose connective tissue from each mouse and examined
for live parasitic stages by light microscopy interference contract (NIC) illumination.
using Nomarski
RESULTS
of
Parasites were not found in histological sections or squash preparations prior to 8 DPI. However, from 8 to 60 DPI, developmental stages of C. bigetzetku were found in fibroblast-like cells of the connective tissues of the cheek. nose, scrotum and less frequently the tongue of all inoculated mice. Mice inoculated orally with a mixture of oocysts and sporocysts appeared subjectively to have larger numbers of parasites than did mice inoculated only with oocysts. Mice inoculated IP with sporozoites alone appeared to have only a moderate number of parasites in the connective tissues. Infected tissues were noticeably swotien at 8-l 6 DPI and were heavily i~ltrated with in~ammatory cells. ~xtraint~tina~ stages. The dimensions and kinetics of each developmental stage of C. bigenetica detected in the histological sections are summarized in Table 1. Qpe I meronts and merozoites. Type I meronts were observed in histological sections at 8 DPI (Fig. 1) and were infrequently encountered. The meronts were spherical or ovoid in shape and contained X- 12 (modes equally of 8 and 10) merozoites. An indistinct, spherical or ovoid ~ytopIasmic mass was usually present with dimensions of 2.8 X 2.7 ‘urn (2.0-4.0 X 2.0-3.0 pm), from which the merozoites appeared to arise by ectomerogony. Usually one (but occasionally two) meront was present within the same host cell. Each meront was located within a spherical or ovoid parasitophorous vacuole. Type I merozoites were elongate, lying parallel within the parasitophorous vacuole and contained a centrally located nucleus and no refractile bodies. 7&e II rneronts atld merozoites. Type I1 meronts were observed in histological sections at if) DPI (Fig.
Sites
infection.
TABLE I--UIMENSIOF;S (IF. FITI) ANIITIMES OP APPEARANCEUC DEVELOPMENTALSTAGI,S OF
C’qosporu bigenerica~u EXPERIMENTALLY INFECTEDCD-l ~tct
Developmental
stage
Type I meronts Type I merozoites Type II meronts Tvpe II merozoites Type III meronts Type 111merozoites Microgametocytes Macrogametes ~n~poruIated oocysts Sporulated oocysts Sporocysts Sporozoites Residuum Spororoites in caryocvsts
Days post-inocuiation
8 10 I0 IU 10 IO, 12
IO.12 IO-14 12.14
12-I-I
Dimension (length~width) (n = 25) 7.5 (iO.O-6.0) x 4.7 (3.5-5.0) X 12.6 (9.0-17.0) x 4.3 (4.0-5.0) x 9.5 (7.0-10.5) x .i.h (3.0-4.2) X 6.2 (4.0~8.0) x 9.5 (7.0-I I .O) x 10.9 (9.G IX) 8.4 (7.5595) 7.1 (6.0-9.0)
6.7 0.9 9.8 1 .o 8.3 1.0 5.x
9.3
(5.0-8.0) (0.X-1.2) (7.0-12.0) (0.X-1.2) (6.0-10.0) (0.X-1.2) (4.0-7.0) (7.0-l 1.0)
x IO.6 (W-l 2.0) x x
7.6 (6.0-X.0) 1.5 (1.2-2.1))
6.5 (5.0-8.0) x 4.1 (3.0-5.0) 5.9 (5.0-7.0) x I .o (I .s-7.0)
Development of C. bigenetica
2) and were more numerous than type I meronts. The type II meronts were spherical or ovoid in shape, larger in size than type I meronts and contained 22-28 (mode = 24) merozoites. The meronts usually contained an indistinct, spherical or ovoid cytoplasmic mass with dimensions of 3.4 X 3.0pm (2.0-6.0 X 2.0-5.0 pm) from which the merozoites appeared to arise by ectomerogony. Usually one (rarely two) type II meronts were present within the same host cell within a spherical or ovoid parasitophorous vacuole. Type II merozoites were moderately elongate and contained a centrally located nucleus and no refractile bodies. Type III meronts and merozoites. Type III meronts were observed in histological sections at 10 DPI (Fig. 3) and were by far the most numerous of all asexual stages seen. They were therefore regarded as the oldest or third asexual generation. These meronts were spherical or ovoid and contained 12-18 (mode = 16) merozoites. An indistinct, spherical or ovoid cytoplasmic mass was usually present measuring 2.0 X 1.9pm (1.0-4.0 X l.O-3.0pm) in size from which the merozoites appeared to arise by ectomerogony. Usually one (but up to three) meronts were found within the same host cell and each was located within a spherical or ovoid parasitophorous vacuole. Type III merozoites were short and stout and contained a centro-posteriorly located nucleus and no refractile bodies. Microgametocytes. Microgametocytes were found as early as 10 DPI, but were most numerous at 12 DPI (Fig. 4). They occurred singly or in groups of up to three and were frequently encountered in areas of connective tissue that contained macrogametes. Microgametocytes were spherical or ovoid in shape and contained 8-14 (mode = 10) microgametes surrounding a spherical or ovoid residuum and lay within a distinct parasitophorous vacuole. Macrogametes. Macrogametes were found as early as 10 DPI. but were most frequently encountered at 12 DPI. The cytoplasm of the macrogamont was lightly granular (Fig. 5) although the size and granularity of these stages was observed to increase with maturity. When fully mature, macrogametes were spherical or ovoid in shape and contained numerous small, cytoplasmic granules, a large prominent nucleus and were located within a distinct parasitophorous vacuole (Fig. 6). Oocysts. Unsporulated oocysts were observed at lo-14 DPI (Fig. 7). The oocyst had a granular cytoplasm and contained one to many homogenous eosinophilic globules. As development proceeded, the sporont contracted and eight sporozoites were eventually formed within the oocyst (Figs. 8 and 9). Sporogony occurred within the sporont membrane and without the formation of a true sporocyst wall, Stieda or substieda bodies. Fully sporulated oocysts were observed at 12 and 14 DPI and had a thin oocyst wall that was observed only in squash preparations. Sporozoites lay parallel to one another and were
17
partially twisted in a semi-spiral around an ovoid, granular residuum. The sporozoites each had a centrally located nucleus and two refractile eosinophilic bodies, each located at either side of the nucleus. Caryocysts. Free sporozoites and caryocysts were first seen at 12 DPI and the caryocysts persisted throughout the duration of the study. Initially, sporozoites separated from the sporocyst residuum, became scattered within the oocyst and finally crossed through the thin oocyst wall (Fig. 10). Sporozoites in caryocysts (Figs. 11 and 12) were within a parasitophorous vacuole and were usually arranged parallel to the long axis of the host cell. Host cells measured 11.1 X 9.9 pm (9.0-14.0 X 8.0-13.0 pm) in size and contained a crescent-horseshoe shaped nucleus usually indicative of macrophages. Caryocysts normally contained a single sporozoite, however, up to four sporozoites were sometimes observed within the same infected host cell. The margin of the host cell usually appeared thicker and darker than the enclosed cytoplasm. Infections with oocysts derivedfrom A. c. mokasen. Examination of mouse tissues at 10,12,14,40 and 60 DPI with oocysts from A. c. mokasen (Figs. 13 and 14) revealed developmental stages of C. bigenetica similar to those found in mice infected with isolates from C. adamanteus. The oocysts were thin walled (Fig. 15) and the sporozoites in fully sporulated oocysts surrounded an ovoid residuum but a true sporocyst wall, Stieda and substieda bodies were not present (Fig. 16). By 40 DPI, however, one of the refractile bodies had disappeared (Figs. 17 and 18) while the larger of the two refractile bodies remained intact. DISCUSSION
Results of our experimental inoculations of mice with oocysts of C. bigenetica are similar, but not identical, to results obtained by Wacha & Christiansen (1982). The present study reports, for the first time, three generations of merogony prior to gamogony in mice inoculated with oocysts. The failure of Wacha & Christiansen (1982) to find asexual stages may best be explained by the fact that they only used 6.0 X 10’ oocysts as inoculating doses whereas we used at least 50 times this number. In addition, we found the times of appearance of gametes and sporulated oocysts to be delayed by approx. 48 h than reported previously. This difference may be explained by the differences in host strain, strain of parasite, numbers of parasites used for experimental inoculations, environmental conditions under which the experiments were conducted or other undetermined factors. However, we are confident that the isolates used in the present study were C. bigenetica. Not only were the isolates found in crotalids, but the oocysts, sporocysts and sporozoites are morphologically indistinguishable from those reported by Wacha & Christiansen (1982).
S. J. UPTON and S. M. BARNARD
18
FIGis. 1-12.
Brightfield photomicrographs experimentally infected FE.
of developmental stages of Caryospora bigeneticrr in the cheek CD-I mice. I-Iematoxylin and eosin stain. (Scale bar = 5 pm.)
I. Type I meront, 8 DPI. Notemerozoites FIG. 2. Type II meront,
10 DPI.
Pm. 3. Type III meront.
10 DPI.
Fro. 4. Micr~~gamet(~cyte,
12 DPI.
Fro. 6. Macrogamete,
12 DPI.
FIG. 7. LJnsporulated FICX. 8 and 9. Sporulated
oocysts,
12 DPI. Note refractile
FIG. 10. Sporulated PK. 1 I. Caryocyst FIG.
12. Caryocyst
(mz).
12 DPI. Note microgametes
Fro. 5. Macrogamont,
oocyst,
(ct), with longitudinalview (ct), with end-on-view
(mi).
12 DPI.
bodies (rb), sporozoites
oocyst with dispersed
dermis
sporozoites ofsporozoite of sporozoite
(sp), and sporocyst
(sp), 14 DPI. (sp), 16 DPI. (sp),
i 6 DPI.
residuum
(sr).
of
Development
of C. bigenetica
19
FIGS. 13-18. Nomarski interference contrast photomicrographs of Catyospora bigeneticu. FIGS. 13 and 14. Oocysts derived from the feces of Agkisfrodon contortrixmokasen. FIGS. 15-l 8. Developmental stages in cheek connective tissues of experimentally infected Swiss-Webster mice. (Scale bar = 5~ m.) FIG. 13. Sporulated FIG. 14. Sporulated FIG. 15. Unsporulated
oocyst, longitudinal
oocyst with refractile
FIG. 16. Sporulated Frc. 17 and 18. Caryocysts
view. Note oocyst wall (ow).
oocyst, end-on-view.Note
oocyst with granular with sporozoites
polar granule(pg).
globule (rg), large nucleus (n), and thin oocyst wall (ow), 10 DPI sporocyst
residuum
(sr) and sporozoites
(sp), each with a nucleus (n) and a refractile
The two species, C. bigenetica and C. simplex, appear to be closely related on the basis of their morphological and developmental characteristics. However, we have noted several differences which could be used to distinguish between the two species. Oocysts of C. bigenetica derived from snakes appear to have a slightly thinner wall than oocysts of C. simplex. The Stieda body of C. bigenetica also appears to be slightly smaller than that of C. simplex and, more distinctively, the positions of the refractile bodies in each species are different; those of C. bigenetica being anterior and central while in C. simplex they are central and posterior (cf. Upton, Current & Barnard, 1986). Pathologically, we have noted that the cheeks of mice appear to be more heavily infiltrated with inflammatory cells and are considerably more swollen during C. bigeneticu infections, even when infections are compared using
(sp), 14 DPI. body (rb), 40 DPI.
low doses (5.0 X 10J oocysts) of C. bigenetica vs. big: doses (2.5 X 105) of C. simplex (unpublished data). Finally, Upton and Barnard (1986) have shown that oocysts of C. simplex readily infect Palestine vipers, Viper-a xanthina palestinae. Although attempts to infect North American crotalids with C. simplex were not completed nor presented in the report, we have since found that eastern diamondback rattlesnakes, C. adamanteus, fail to become infected with oocysts of C. simplex (unpublished data). Thus, C. bigenetica and C. simplex appear to represent two distinct species. Wacha & Christiansen (1982) reported two types of meronts in snakes. Type I meronts which contained 12 (10-14) merozoites and a residuum and type II meronts containing seven (four to nine) merozoites that lay either parallel or in a staggered formation within the parasitophorous vacuole with no observ-
20
S. J. UPTON and S. M. BAKNARD
able residuum. In the present study, three types of meronts were seen. At 8 DPI, type I meronts were present and contained 8-12 slender, parallel merozoites. At IO DPI, large type II meronts, with about 22-28 merozoites and the highly numerous type II1 meronts, with about 12-18 merozoites were seen. Comparing data from the two studies, we are unable to ascertain whether the two types of meronts found in the intestine of snakes correspond to any of the meronts we observed. It is possible that no retationship exists between the different generations of meronts that occur in the different hosts. By 8 DPI, we noted that all mice inoculated with oocysts appeared lethargic and their facial tissues, tongue and scrotum appeared edematous. Wacha & Christiansen (1982) noted a similar condition in the facial tissues of mice they infected. However, two of the five mice they inoculated died on about 10 DPI whereas we have never observed any mortality due to C. bigeneticu in over 100 mice inoculated with various doses over the last 2 years, including CD- 1, BALB/c, CS7BL/6, and Swiss-Webster mice. Since our previous work (Upton et al., 1984), we have inoculated additional mice with C. simplex and have found that this coccidium also develops in the scrotal dermis of mice. The finding that both C. simplex and C. bige~etjca develop in facial, tongue, and scrotal connective tissues suggests the selection of these predilection sites may be temperaturedependent. These areas tend to be slightly cooler regions of the body. Although infections are observably uncomfortable for mice, the location of parasites in these sites may allow for ready access to intestinal cells in the snake during the early stages of digestion. Marcone (1908) reported subcut~eous nodular coccidiosis from a dog from Italy and he named this coccidian “Coccidium nudum”. Shelton, Kinter & MacKintosh (1968) and Sangster, Styler & Hall (1985) also reported similar lesions from dogs in Missouri and Georgia, U.S.A., respectively. All three of these reports not only describe dermal meronts, but Eimeriidae/Sarcocystidae-like macrogametes and microgametocytes as well. It is interesting to note that
the dog studied by Shelton et al. (1968) died 2 months later of acute canine distemper, a virus reputed to cause immunodepression in canids. The fact that C. simplex and C. bigenetica both develop in the intestinal tract of poikilotherms (snakes) and, experimentally, in the connective tissues of homeotherms (rodents) suggests that, under the appropriate conditions, both species (and perhaps other Caryospwu spp. as well) may have the potential to become nonhost specific in immunosuppressed hosts. if this is the case, it is possible that the Curyuspora may represent the causative agents of canine subcutaneous nodular coccidiosis.
MARCONE G.
REFERENCES 1908. Sporozoen-Dermatosen des Hundes.
~eitschri~ fiir Infektionskrurzkheiten, Purus~te~krz~nkheiten z&d ~~ie~~ der Haustiere 4: 5-32. SANGSTER
Coccidia
L.T..
STYLER E. L. oi HALL G. A. 19%. associated with cutaneous nodules in a dog.
Veterinary Pathology 22: SHELTON
186-188.
G. C., KINTER L. D. & MACKINTOSH D. 0. 1968.
A coccidia-like organism associated with subcutaneous granulomata in dog. Journul of’the American Veterinag
MediculAssociation TIUHUN
152: 263-267.
G. & STOCKDALE P. I-1. G. 1982. Sensitivity
and specificity of the indirect fluorescent antibody test rn the study of four murine coccidia. Joz~r~a~of ~r~f~~aoZ~,~~
29: 129-132. ~JPTON S. J., CURRENT W. L., J.5~~ls-rJ. V. &BARNARD S. M.
1984. Extraintestinal development of C’uryosporu sbnpke.w (Apicomplexa:Eimeriidae) in experimentally infected mice, Mus musczdzrs. Joztrrzal of t’rotozoolog_v3 1: 3X398. UPTOK S. J. & BARNARD S. M. 1986. Experimental transof simplex mission Cff ~osporcr (Apicomplexa: Eimeriidae) to Palestine vipers, t’$rt-44 ,~atzt~zitz~z palestinae (Serpentes: Viperidae). Jozrrnai of Proio-
;oologv 33: 129-130. UPTON S. J., CURRENT W. L. s( BARNARD S. M. 19X6. A review of the genus Curyospora (Apicomplexa:limeriidae). Systematic Parasitology 8: 3-2 1. Wacw, R. S. & CHRISTIANSEN J. L. 19X2. Development of Chtyospom bigeneticu n. sp. (Apicomplexa:Eimeriidae) in rattlesnakes and laboratory mice. Jownal of‘ frotozoolop 29: 272-278.