ANTIGENIC ANALYSIS OF GAMONTS OF HEPATOZOON CANIS PURIFIED FROM LEUKOCYTES

Antigenic Analysis of Gamonts of Hepatozoon canis Purified from LeukocytesAuthor(s): Gad Baneth, Varda Shkap, Michael Samish and Charles L. JaffeSource: The Journal of Parasitology, Vol. 86, No. 2 (Apr., 2000), pp. 289-294Published by: The American Society of ParasitologistsStable URL: http://www.jstor.org/stable/3284771 .

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J. Parasitol., 86(2), 2000 p. 289-294 ? American Society of Parasitologists 2000

ANTIGENIC ANALYSIS OF GAMONTS OF HEPATOZOON CANIS PURIFIED FROM LEUKOCYTES

Gad Baneth, Varda Shkap*, Michael Samish*, and Charles L. Jaffet School of Veterinary Medicine, Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel

ABSTRACT: Hepatozoon canis is a tick-borne apicomplexan parasite of dogs that infects neutrophils and parenchymal tissues. To study the antigenic characteristics of this parasite, a technique was devised for the purification of gamonts from peripheral blood neutrophils. White blood cells were separated on Ficoll-Hypaque density gradients and the gamonts were released from the host neutrophils by nitrogen cavitation. The blood used for purification originated from dogs with natural or experimental infections of H. canis with a parasitemia of 1.4-33%. The number of parasites collected ranged from 1.5 X 106 to 4.2 X 107. Portions of purified gamonts were separated and examined under phase and scanning electron microscopy, and the remaining purified parasites were then used as a source of antigens to characterize the humoral immune response by western blot analysis. Serum antibodies from infected dogs recognized more than 15 gamont antigens, and the antigenic patterns observed with sera from naturally and experimentally infected dogs were nearly similar. Four immunodominant protein bands of relative molecular weights of 107, 88, 63, and 28 kDa were recognized by all of the sera examined. The technique applied here for the isolation of host cell-free gamonts will facilitate studies on antigenic composition and immune responses against H. canis and on antigenic relationships between Hepatozoon from different host species and geographic regions.

Hepatozoon canis is an apicomplexan protozoan parasite of dogs that is transmitted by ticks (James, 1905; Christophers, 1907). Canine hepatozoonosis has been reported from tropical, subtropical, and temperate regions of all continents except Aus- tralia (Craig, 1990). Hepatozoonosis in dogs may vary from subclinical infections with low parasitemia, to a severe and life- threatening disease in young puppies or immunosuppressed animals (Hervas et al., 1995; Baneth and Weigler, 1997). A different form of canine hepatozoonosis is caused by Hepatozoon americanum, a protozoon with morphologically similar gamonts in leukocytes that induces a severe myositis with gait ab- normalities in dogs in the southern USA (Vincent-Johnson et al., 1997). Infection with H. canis occurs when dogs ingest a tick containing mature oocysts. Sporozoites released in the gut of the dog penetrate the intestinal wall and disseminate via the blood or lymph to parenchymal organs such as the liver, spleen, and bone marrow, where merogony takes place. The released meronts then penetrate neutrophils and form gamonts that can be detected in the peripheral blood. The parasite life cycle is completed when a tick ingests infected dog blood containing gamonts, followed by formation of oocysts in the tick hemo- coel.

Antibodies to H. canis gamonts, detected by an indirect fluorescent antibody test (IFAT), have been described in naturally and experimentally infected dogs (Shkap et al., 1994; Baneth, Shkap et al., 1998). In Israel, seroprevalence is high (33%), and 1% of the seropositive dogs had gamonts in their circulating neutrophils (Baneth et al., 1996). Whereas most infected dogs have relatively low parasitemia of 1-2%, about 10% of the infections in Israel are diagnosed as having high parasitemia with 40-100% infected neutrophils in the peripheral blood, of- ten accompanied by clinical manifestations of fever, severe leth- argy, and anemia. Some of these latter dogs develop neutro- philia that may reach 90,000 neutrophils/[l of blood and result in the presence of 8,000-90,000 gamonts/pl of blood (Baneth

and Weigler, 1997). In the present study, a simple method for

harvesting H. canis gamonts from peripheral blood neutrophils by density gradient centrifugation and nitrogen cavitation was developed. For the first time, antigens of host cell-free gamonts were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by western blotting using sera from naturally or experimentally infected dogs.

MATERIALS AND METHODS Source of H. canis and purification of gamonts

Heparinized peripheral blood (82 ml) was obtained by venipuncture from 5 dogs, of which 4 were naturally infected and 1 experimentally infected with H. canis, 62 days postinfection. The infected blood was layered onto test tubes with a sintered filter (Uni-Sepl0+, Novamed, Jerusalem, Israel) that prevents mixing of the blood and the Ficoll- Hypaque density-gradient medium. After centrifugation at 800 g for 20 min at room temperature, a band containing leukocytes was collected, suspended in 30 ml of phosphate-buffered saline (PBS) pH 7.2, and washed 3 times by centrifugation at 800 g for 20 min with PBS. The leukocytes were then resuspended in 30 ml of PBS, equilibrated in a nitrogen cavitation chamber at 500 psi for 10 min, and disrupted by releasing the pressure (Hunter and Commerford, 1961). The material containing cell-free gamonts and debris was collected in a centrifuge tube and centrifuged for 10 min at 800 g. The pellet containing purified parasites was resuspended in PBS and washed 3 times by centrifugation at 800 g for 20 min at 4 C. The final pellet containing released gamonts was resuspended in a minimal volume of PBS (0.5-3 ml), and the number of purified parasites was determined in a Neubauer hemocytometer with 0.5% trypan blue. At each stage of purification the material was examined by Nomarski phase microscopy. Aliquots of the purified and counted gamonts were taken and frozen at -70 C.

The yield of gamonts was determined as follows. For each dog, a separate sample (2 ml) of the original venous blood was collected in an EDTA tube, and a complete blood count was performed. The percentages of neutrophils and of parasitized neutrophils were determined from Giemsa-stained blood smears, and the parasitemia percentages were calculated (parasitemia percentage = infected cells per 100 neutrophils). The number of parasites/pi blood (parasitemia percentage X

neutrophils/il blood) and total number of parasites (ml of blood used X gamonts/ml blood) for each isolation was estimated. The yield of gamonts from each purification was then calculated by dividing the total number of purified parasites by the total number of parasites in the original blood sample.

Antibodies Positive serum samples were obtained from experimentally (n = 3)

and naturally (n = 3) infected dogs. Negative control sera were obtained

Received 11 May 1999; revised 31 August 1999; accepted 1 Septem- ber 1999.

* Department of Parasitology, Kimron Veterinary Institute, Bet Dagan, Israel.

t Department of Parasitology, Hebrew University-Hadassah Medical School, Jerusalem, Israel.

289



290 THE JOURNAL OF PARASITOLOGY, VOL. 86, NO. 2, APRIL 2000

from tick-free, laboratory-raised dogs prior to infection with H. canis. Experimentally infected dogs were inoculated with H. canis as previously described (Baneth, Shkap et al., 1998). Briefly, 3-mo-old laboratory-raised dogs were inoculated with 30 adult Ripicephalus sangui- neus ticks, repleted as nymphs on a naturally infected dog. The dogs developed hepatozoonosis with parasites that were identified in blood smears and bone marrow aspirates by light microscopy. Sera from naturally infected dogs originated from animals diagnosed with hepatozoonosis at the Veterinary Hospital of the Hebrew University School of Veterinary Medicine. These dogs exhibited clinical symptoms of leth- argy or anemia and gamonts of H. canis were detected in their blood.

IFAT

Serum samples were analyzed by IFAT for IgG antibodies against H. canis, as described previously (Shkap et al., 1994). Antigen slides were prepared from the blood of a naturally infected dog with high parasitemia. The buffy coat was washed with PBS by centrifugation 3 times, and the final pellet was resuspended in a mixture of equal volumes of PBS and 3% bovine serum albumin (BSA). Thin blood smears were made on glass slides and dried at room temperature. The slides were immersed for 10 min in acetone and then stored at -80 C until used. Before use, antigen slides were warmed and dried at 37 C for 30 min. A series of successive 2-fold dilutions of serum in PBS, from 1:16 to 1:4,096, was applied to the smears and incubated for 30 min at 37 C in a humid chamber. The slides were washed 3 times in PBS and blotted dry. Rabbit anti-dog IgG fluorescein conjugate was applied to the wells at 1:70 dilution. The slides were then incubated at 37 C for 30 min, washed as before, and dried. The smears were mounted under coverslips in PBS-buffered glycerol (pH 8.5) and examined under a fluorescent microscope.

SDS-PAGE and western blot analysis

The frozen suspension of purified gamonts from H. canis isolate no. 2548 was thawed at room temperature, and after a brief spin the protein concentration of the supernatant was determined by using the Bradford method (Bradford, 1976). Gamonts were further solubilized in sample buffer (0.025 M Tris-glycine, pH 6.8; 2% w/v SDS; 15% w/v glycerol and bromphenol blue) at 100 C for 3 min. The gamont antigen at 10 p[g protein/lane was subjected to SDS-PAGE (7.5-17.5% gradient gel) under nonreducing conditions (Laemmli, 1970). The resolved polypep- tides were transferred overnight onto nitrocellulose membranes in Tris- glycine buffer containing 20% methanol at constant 35 mA and 4 C (Towbin et al., 1979). The membranes were blocked with PBS containing 3% casein for 2 hr at room temperature, followed by 5 successive washings with PBS containing 0.5% Tween 20 (PT). Dog sera were diluted in PT, applied to individual strips, and incubated overnight at 4 C. The nitrocellulose strips were washed 7 times as before, and the bound antibody was detected with rabbit anti-dog IgG conjugated to horseradish peroxidase at 1:3,000 dilution in PBS containing 0.2% BSA. After incubation for 1 hr at 37 C the nitrocellulose strips were washed 8 times with PT and developed with 0.005% 3-3'-diamino- benzidine (DAB, Sigma, St. Louis, Missouri) supplemented with 0.03% cobalt chloride and 0.3% H202.

The reactions of positive and negative control sera on noninfected canine leukocytes were also examined. Blood from a laboratory beagle that had been shown to be negative for parasites by microscopic examination and for anti-H. canis antibodies by IFAT was separated by Ficoll-Hypaque, as described above, and frozen. For western blot analysis the leukocytes were thawed, solubilized in a SDS-PAGE sample

buffer, electrophoresed, and blotted as described above. The membranes were reacted with pre- and postinfection sera from an experimentally infected dog.

Scanning electron microscopy (SEM)

For SEM, purified gamonts in PBS were applied onto round glass coverslips and fixed with 1% glutaraldehyde for 2 hr at 4 C. The preparations were washed 3 times with PBS, twice in 50% ethanol for 30 min, and transferred to 70% ethanol overnight. The slides were dehy-

drated in 95% and then 100% ethanol, dried at 42 C, and covered with pure gold on a rotatory turntable high-vacuum unit (Klainer and Betsch, 1970). The samples were examined under a GOUL-35C SEM at 20 kV.

RESULTS

Cell-free preparation of gamonts

Purification of H. canis gamonts was carried out from blood with parasitemia ranging from 1.4% to 33%. The number of purified parasites collected ranged from 1.5 X 106 to 4.2 X 107, and the yield of purified gamonts ranged from 3.6 to 24%.

Phase microscopy of partially purified parasites suspended in PBS after disruption by nitrogen cavitation and washes in PBS (Fig. 1) showed morphologically intact gamonts with minimal cell debris contamination. High-power Nomarski phase microscopy of parasitized leukocytes prior to cell disruption depicted elongated ellipsoidal gamonts located in the centers of the host cells, each having a thick capsule separating it from the host cell cytoplasm (Fig. 2a). Gamonts isolated from cells after ni-

trogen cavitation still retained the external capsule seen prior to disruption and maintained their external ellipsoidal shape (Fig. 2b). The isolated gamonts appeared to have an internal hooklike shape at 1 of their poles that had not been observed prior to cell breakage.

SEM of the purified gamonts showed an ellipsoidal organism contained in an electron-dense capsule and a lucent outer membrane (Fig. 3). A conoid structure bulges cranially from the clearly defined border of the electron-dense capsule and is en- closed within the outer membrane that envelops the whole parasite. No cellular organelles or parts that could clearly be attri- buted to the host cells were observed to be attached to the 20 isolated gamonts that were observed by SEM.

Serology and western blot

All experimentally infected dogs developed high IFAT antibody titers to H. canis; these ranged from 1:256 to 1:1,024 in tests performed on serum samples taken on days 41-60 after the dogs had been inoculated with the infected replete ticks. Similar antibody reactivity was observed with sera from naturally infected dogs, except for 1 that exhibited an unusually high titer of 1:4,096.

Western blot analysis revealed multiple bands when the gamont antigens were reacted with sera from either experimentally or naturally infected dogs (Figs. 4, 5, respectively). The proteins recognized by the immune sera were of relative molecular

weights ranging from 10 to 150 kDa. Minimal diffuse and weak nonspecific staining was observed with the control preinfection sera (Fig. 4, lanes 2, 4, and 6). Four, strongly stained immunodominant bands of 107, 88, 63, and 28 kDa and 5 other weakly stained bands of 120, 92, 72, 21, and 17 kDa were recognized by sera from all of the infected dogs. All of the sera from infected dogs, excluding dog no. 4 (Fig. 5, lane 3), also reacted with the 123- and 1 12-kDa bands. Sera from all the experimentally infected dogs and from 1 naturally infected dog (Fig. 5, lane 4) recognized an antigen at 19 kDa. In addition to the immunodominant parasite antigens, several antigens, such as the 29-kDa band, appeared to react only with individual dog sera (Fig. 4, lane 3). Weakly staining bands ?14 kDa that were not uniform in either number or molecular weight were identified by sera from the various dogs. Serum from experimentally infected dog no. 1 (Fig. 4, lane 1) reacted with 2 bands of 93 and 23 kDa when tested with the preparation of leukocytes from a healthy dog.



BANETH ET AL.-ANTIGENIC

ANALYSIS OF H. CANIS GAMONTS 291

o4z A UP) Ni

f- Z

At %MW z ? "Ot ..v

gj?:x Filtt. ON -,Vv, qvy, Z-g- Zfg T-Px-

L? x-E X UO. RI

A MCZ

so NO= I

kS ?-KTJMMP *tT Lng- 4R

IV 101?;?Z-,..-.!?Ml'tRl- Yz ...... ...

....... ...... FIGURE 1. Light microscopy of partially purified Hepatozoon canis gamonts following separation from peripheral blood and leukocyte disrup-

tion by nitrogen cavitation. Bar = 50 prm.

DISCUSSION

This study involved the isolation and harvesting of H. canis gamonts from whole peripheral blood in the first attempt to

produce leukocyte-free H. canis gamonts for antigenic characterization. Several techniques have been used previously to free

hemoparasites from their host cells, most of them relied on the differences in physical or chemical properties between the cell and the parasite to facilitate disruption of the host cell without destruction of the intracellular pathogen.

Nitrogen cavitation has been used to obtain host-free hemo-

parasites from the blood of different animal hosts, i.e., Plas- modium knowelsi (Trigg et al., 1970) and Plasmodium falciparum (Williamson and Cover, 1975) from simian and human blood, respectively, and Theileria sergenti (Shimizu et al., 1988) and Babesia ovata (Shimizu et al., 1992) from bovine red blood cells. Unlike infections with other hemoparasites in which several different stages of the parasite may be present simultaneously in the same peripheral blood cells (Williamson and Cover, 1975), in H. canis infection, only gamonts are detected in peripheral blood smears of infected dogs. Therefore, the harvested H. canis preparations comprise only gamonts and can be considered homogenous, in contrast to some other he- moparasite preparations made by this method.

The application of nitrogen cavitation, a relatively simple technique, to the purification of H. canis resulted in efficient purification of morphologically intact gamonts with minimal cell debris contamination, and facilitated antigenic studies on cell-free parasites. From the SEM images, it does not appear that there was severe external damage to the gamonts, as they retained their characteristic shape. On the other hand, the formation of the hook-shaped gamonts, revealed by phase micros-

copy, may be a result of either damage to internal parasite struc- tures or disruption of links between the neutrophil cytoskeleton

and the parasite. Alternatively, this transformation may mimic

changes that take place when the gamont is released from a

neutrophil ingested by the vector tick. A curved posterior end has been reported from other species of Hepatozoon including Hepatozoon lygosomarum (Allison and Desser, 1981) and may be a morphologic characteristic of these species or related to their containment within the host blood cell.

The number of parasites purified depended on the number of infected neutrophils and the successful disruption of infected cells with the preservation of intact gamonts. The yield of purified gamonts was 524% in all of the purifications performed, and because there is no evidence from microscopic examination that disruption of the infected neutrophils was incomplete under the conditions employed, some of the gamonts were probably destroyed by the nitrogen cavitation. However, at least 1.5 mil- lion gamonts were isolated from every harvest, even when the

parasitemia was only 1.4%. This quantity of parasites was suf- ficient for analysis of H. canis antigens by western blotting. This method may also be useful for isolating antigens from the blood of animals infected by related parasites that cause low

parasitemias, such as H. americanum (Macintire et al., 1997; Vincent-Johnson et al., 1997) and Hepatozoon species in cats

(Baneth, Aroch et al., 1998; Beaufils et al., 1998). In previous studies, we demonstrated that H. canis gamonts

are specifically recognized by sera from both naturally and ex-

perimentally infected dogs (Shkap et al., 1994; Baneth, Shkap et al., 1998). In the present study, we describe for the first time the gamont antigens recognized by immune sera using western

immunoblotting techniques. Infected dog sera reacted with at least 10 gamont molecules. Little difference was seen between sera from naturally and experimentally infected dogs in their

recognition of the major antigenic bands. Indeed, sera from all 6 dogs examined reacted with 9 common antigens (120, 107,




I. .. i,

~Mal

FIGURE 2. Nomarski phase microscopy of Hepatozoon canis gamonts in cells before and after disruption by nitrogen cavitation, a. An infected neutrophil containing a gamont. b. A gamont following nitrogen cavitation and removal of cell debris by centrifugation. Bar = 10 [tm. Note the centrally located gamont in the cytoplasm of the neutrophil compressing the lobulated host cell nucleus (2a).

92, 88, 72, 63, 28, 21, and 17 kDa), whereas 5 out of 6 dogs reacted with 2 additional antigens (123 and 112 kDa), indicat- ing the similarity of the individual host responses to H. canis gamonts antigens. The recognition of the 19-kDa band by the experimentally infected animals and by only 1 naturally infected dog may stem from the fact that all of these dogs were inoculated with the same isolate of H. canis, whereas naturally infected dogs could be infected with several antigenically dis- tinct field strains. The western blotting patterns for the individual dogs were very similar for antigens with molecular weights

>63,000, whereas the reaction against the lower molecular weight bands varied considerably.

It is expected that dogs in the early stages of infection, before the appearance of gamonts in their peripheral blood, may show seroreactivity with fewer antigenic bands revealed by western blotting than dogs with parasitemia, such as those described here. This should allow the identification of antigens shared by gamonts and other stages of the parasite. In a previous study, we have shown that anti-gamont antibodies could be detected by IFAT in experimentally infected dogs, even before gamonts

.

u

FIGURE 3. Scanning electron micrograph of an Hepatozoon canis gamont freed from a neutrophil by nitrogen cavitation. Note the conoid structure (enlarged) protruding from the electron-dense inner membrane and extending cranially to the outer limiting membrane. Bars =1 p~m.



BANETH ET AL.-ANTIGENIC ANALYSIS OF H. CANIS GAMONTS 293

M 1 234 5 67 k

106.0 - 107 kDa

92.5 - .- 88kDa 68.0-

S63 kDa 46.0 -

30.0 - 30.0..4

28 kDa

14.0 -

FIGURE 4. Western blot analysis of Hepatozoon canis gamont antigens recognized by sera from experimentally infected dogs. Preinfection sera: dog 1, lane 2; dog 2, lane 4; and dog 3, lane 6. Postinfection sera: dog 1, lane 3; dog 2, lane 5; and dog 3, lane 7. Reaction of postinfection serum from dog no. 1 with uninfected canine buffy-coat is shown in lane 1. Molecular weight markers are shown in lane M. Note the 28-, 63-, 88-, and 107-kDa immunodominant bands marked with arrows.

appeared in the peripheral blood (Baneth, Shkap et al., 1998). We suggested that those antibodies were generated against conserved epitopes found in earlier tissue stages of the parasite.

The reactivity of noninfected leukocytes with sera from an experimentally infected dog is an interesting finding that may

be explained by cross-reactivity against a conserved epitope on a host antigen or perhaps by the formation of autoantibodies against leukocytes. The partial purification of H. canis gamonts enabled us to identify a number of immunodominant parasite antigens that were recognized by the immune system of the

M 1 2 3 4 5

106.0 -107 kDa

92.5- 88 kDa 68.0 -

63 kDa

46.0 - 30.0

--.- 28 kDa

14.0 o

,1.

FIGURE 5. Western blot analysis of Hepatozoon canis gamont antigens recognized by sera from naturally infected dogs. Reaction with sera from naturally infected dogs nos. 4, 5, and 6 are shown in lanes 3, 4, and 5, respectively. Incubation with pre- and postinfection sera from an experimentally infected dog (no. 3) in lanes 1 and 2, respectively. Molecular weight markers, lane M. Note the 28-, 63-, 88-, and 107-kDa immunodominant bands marked with arrows.




infected host. Future studies using purified parasites will facilitate comparisons between host humoral and cell-mediated immune responses to gamont antigens during the acute and chron- ic stages of H. canis infection. The development of new diagnostic tools based on a knowledge of defined parasite antigens will facilitate the examination of possible antigenic relationships between Hepatozoon isolates from different geographical regions, different host species, and also from potentially closely related species of apicomplexan parasites. These studies may be strengthened further by morphological characterization and comparative molecular differentiation based on gene sequences.

ACKNOWLEDGMENTS

The authors thank Noam Safran, Ora Eyal, Lea Graziani, I. Savitsky, Evgeni Alekseev, and Itamar Aroch for their assis- tance.

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ANTIGENIC ANALYSIS OF GAMONTS OF HEPATOZOON CANIS PURIFIED FROM LEUKOCYTES

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