Vaccination of pigs with Toxoplasma gondii antigens incorporated in immunostimulating complexes (iscoms)

Vacinação de suínos com antígenos de Toxoplasma gondii incorporados a complexos imunoestimulantes (iscoms)

Vaccination of pigs with Toxoplasma gondii antigens incorporated in immunostimulating complexes (iscoms)

Vacinação de suínos com antígenos de Toxoplasma gondii incorporados a complexos imunoestimulantes (iscoms)

R.L. Freire^I; I.T. Navarro^I; A.P.F.R.L. Bracarense^I; S.M. Gennari^II

^ICentro de Ciências Agrárias da Universidade Estadual de Londrina Caixa Postal 6001 86051-990 - Londrina, PR

^IIFaculdade de Medicina Veterinária e Zootecnia, USP

ABSTRACT

Immunity to Toxoplasma gondii was studied in pigs, after vaccination with T. gondii antigens incorporated into immunostimulating complexes. Nine pigs (group 1 - G1) were inoculated subcutaneously with T. gondii iscoms (LIV-5 sample) and three doses were given at 21 and 13 day-intervals. The results were compared in other three groups of nine pigs each: animals in group 2 (G2) were immunized with the LIV-5 antigens without Quil A, animals in group 3 (G3) were inoculated with tachyzoites of RH T. gondii isolate, and animals in group 4 (G4) received no vaccination. Four animals were neither vaccinated nor challenged with T. gondii (group 5 - G5). Thirty days after vaccination, pigs were challenged orally with 5´10⁴ oocysts at AS-28 T. gondii isolate. Euthanasia was carried out 47 days after challenge and specimens of the heart, muscle, brain, liver, tongue and retina were inoculated into mice. Three out of nine pigs from G2 and one out of nine pigs from G4 showed hypertermia after the challenge. Antibody response was analysed by indirect fluorescent antibody test. The first iscom immunization (G1) induced low antibody levels, the second and third produced high antibody levels, similarly to the RH isolate infection (G3). Western blotting analysis indicated that the antibody response in animals in G1, after challenge, was more intense than in animals in the non-vaccinated group. T. gondii was not isolated by bioassays from tissues of iscom vaccinated pigs, while recovery was obtained from four animals in G4, one in G2 and one in G3.

Keywords:Toxoplasma gondii, pig, vaccine, iscom

RESUMO

A imunidade ao Toxoplasma gondii foi estudada em suínos, após a vacinação com antígenos de T. gondii incorporados a complexos imunoestimulantes (iscoms). Nove animais (grupo 1-G1) foram inoculados, via subcutânea, com três doses de iscoms (amostra LIV-5) em intervalos de 21 e 13 dias. Para comparar os resultados obtidos, outros três grupos com nove suínos cada foram formados: o grupo 2 (G2) foi imunizado com os antígenos da amostra LIV-5 sem o iscom, o grupo 3 (G3) foi inoculado com taquizoítas da amostra RH de T. gondii e o grupo 4 (G4) não foi imunizado. Quatro animais não receberam a vacina nem o desafio com o T. gondii (grupo 5-G5). Trinta dias após as imunizações os suínos foram desafiados, via oral, com 5´10⁴ oocistos da amostra AS-28 de T. gondii. Os animais foram sacrificados 47 dias após o desafio e fragmentos de coração, músculo, cérebro, fígado, língua e retina foram colhidos para a inoculação experimental em camundongos. Três dos nove suínos do G2 e um dos nove suínos do G4 apresentaram temperatura elevada após o desafio. A resposta humoral (IgG) foi analisada pela reação de imunofluorescência indireta. A primeira vacinação com o iscom induziu baixos níveis de anticorpos, a segunda e a terceira resultaram em altos níveis, semelhantes aos encontrados na infecção com a amostra RH (G3). O Western blotting indicou que a resposta humoral do G1, após o desafio, foi mais intensa que a dos animais não vacinados. O T. gondii não foi isolado no bioensaio das amostras de tecido dos suínos vacinados com iscom, mas foram recuperados em quatro suínos do G4, um suíno do G2 e um do G3.

Palavras-chave:Toxoplasma gondii, suíno, vacina, iscom

INTRODUCTION

Toxoplasmosis is one of the most prevalent zoonoses in the world, and in spite of the high frequency of inapparent infections, T. gondii can manifest in a systemic and serious manner, as occurs in the congenital disease and in immune depressed animals (Frenkel, 1990). Transmission to man and other homeothermic animals is mainly transplacental or by ingestion of food contaminated with parasite oocysts or cysts. Oocysts are eliminated by felines, including the domestic cat, and the cysts are present in animal tissues of chronically infected animals (Dubey, Beattie, 1988).

Swine have received special attention from the public health because they are an important reservoir of Toxoplasma for human populations due to the longevity of the tissue cysts, and the wide dissemination of the infection in this animal species (Dubey et al., 1984). Dubey et al. (1991), in a study on the prevalence of anti T. gondii antibodies in swine in several states in the USA, found that about 25% of the herds analyzed had been exposed to the parasite. A quarter of the world swine herd is located in Brazil and toxoplasmosis prevalence varies from 9.6% to 54.12% mainly due to social and cultural habits and the type of management used in swine rearing in each region (Vidotto et al., 1990; Porto et al., 1999; Suaréz-Aranda et al. 2000).

Among strategies to control toxoplasmosis, immunization of domestic animals of economic interest is studied to reduce the fetal damage and the number of tissue cysts in these animals. Vaccination with live samples or dead antigens associated with different adjuvants has been conducted over the years with variable success (Buxton, 1993; Freyre et al., 1993; Dubey et al., 1994). Lunden (1995) used soluble antigens from the RH sample incorporated in immunostimulating complexes (iscoms) in sheep and observed that both the humoral and cellular immune response, assessed after the vaccinations and after the challenge, were significant when compared to the control group.

The aim of this study was to construct immunostimulating complexes (iscoms) with T. gondii tachyzoites surface antigens from a swine sample, to check the humoral immune response, identify the antigens recognized by the immune system of the animals after immunization and to assess the presence of tissue cysts in vaccinated swine after the challenge.

MATERIALS AND METHODS

Forty swine were subdivided into five experimental groups, four groups of nine animals (G1, G2, G3, and G4) and one group containing four animals (G5). The G1 animals were vaccinated on three occasions (experimental days 0, 21^st and 34^th) with the immunostimulating complexes (iscoms). The G2 animals were vaccinated with an inoculum containing only T. gondii tachyzoite surface antigens and lipids, following the same vaccination scheme previous described. The G3 animals received live tachyzoites from the virulent RH sample on two occasions, experimental days 0 and 34^th. The G1, G2 and G3 animals were challenged on the 64^th day. G4 was not vaccinated and received only the challenge dose with 5x10⁴ AS-28 oocysts. G5 was kept as a healthy control group (Fig. 1). Body temperature was taken at four different periods and blood samples were collected every 10 days from experimental day 0 until the euthanasia, which was carried out 47 days after the challenge. Blood and 100g samples from the brain, liver, heart, tongue and skeletal muscle (diaphragm and triceps) were collected from each slaughtered animal and the retinas from the animals in G4 and G5. The carcass was weighed at slaughter.

Three T. gondii strains kept in Swiss mice were used. Sample LIV-5 tachyzoites isolated primarily from swine muscle (Navarro et al., 1992) were used to prepare the Iscom. Tissue cysts from the AS-28 sample (Deane, 1971) obtained from chronically infected mice brain, were used in the experimental infection of cats that produced oocysts for the swine challenge. Tachyzoites were used from the RH sample (Sabin, 1941) to inoculate animals in G3 and, as antigen, for indirect fluorescent antibody test (IFAT).

Forty male Large White ´ Landrace pigs, serologically negative to T. gondii, were used. The animals were acquired at 47 days of age, weighing on average 17.2kg, and the experiment began at day 66.

Female Swiss mice weighing 25 to 30g were used for bioassay and to obtain T. gondii tachyzoites or tissue cysts.

The iscoms were prepared by the method described by Lundén et al. (1993) and Lundén (1995). LIV-5 sample tachyzoites, diluted in phosphate buffered saline, were added to a stock solution of MEGA-10 detergent (20% vol/vol) (Sigma Chemical Co., St. Louis, MD, USA) containing phosphatidylcholine and cholesterol (10mg/ml of each) (Sigma Chemical Co., St. Louis, MD, USA) and a 4:1 protein:cholesterol ratio was obtained. The suspension was incubated for 30 minutes at room temperature and centrifuged at 1,500g/15 min. The supernatant was collected, the adjuvant Quil-A added (Superfos Biosector, Vedbaek, Denmark) and a 1:5 cholesterol:Quil-A ratio was obtained. After homogenization the solution was dialyzed against PBS at room temperature in the first 24 h and then at 4ºC for 48 hours. The protein concentration of the iscoms was determined using the bicinchoninic acid protein assay reagent (Pierce Biotecnology Inc., Rockford, IL, USA) and bovine serum albumin as standard. The iscoms morphology was examined using an electronic transmission microscope (Bomford et al., 1992) with 50,000 and 80,000 magnification. The presence of LIV-5 sample proteins in the iscoms formed was checked by polyacrilamide gel electrophoresis. The total volume of the vaccine was filtered through 0,22µm membrane and stored at -20ºC until use.

The humoral immune response to T. gondii was determined by the indirect fluorescence antibody test (IFAT) according to Camargo (1973). Conjugated goat anti-pig and goat anti-mouse immunoglobulin G were used¹. Negative and positive control serums for the species of interest were used throughout the test. Dilution greater or equal to 1:64 was considered positive (Vidotto et al., 1990).

A lysate of LIV-5 tachyzoites was separated by polyacrilamide gel electrophoresis on 12% slab gels, using the discontinuous sodium dodecyl sulfate (SDS) buffer system described by Laemmli (1970), and transferred to nitrocelulose membrane (pore size 0.45µm) (Bio-Rad Laboratories, Hercules, CA, USA) (Towbin et al. 1979). The Western blotting was carried out according to the protocol used by Breganó (1994). After blocking for one hour with non fat milk at 5% in PBS (0.01 Mol) the membrane was washed and incubated for 2h with the serum, at 1:50 dilution in PBS with 1% non-fat milk. Serum samples from the G1 animals collected on the 56^th experimental day and from G1 and G4 animals collected on the 86^th experimental day were used; serum samples from the G5 animals were used as negative controls. The membrane was incubated with horseradish peroxidade-conjugated rabbit anti-pig IgG¹ diluted 1:2000 in PBS with 1% non fat dry milk for 1h.

Tissue samples collected from the experimental animals were processed and inoculated in Swiss mice to detect the presence of cysts in the studied animals. Brain and liver fragments, as well as the retina from the G4 and G5 animals, were ground individually (Dubey et al., 1995). The other tissues were submitted to peptic digestion according to the protocol described by Dubey (1998b). For each tissue sample processed five Swiss mice were inoculated with a 1ml dose via intraperitoneal. The mice were observed for six weeks and when clinical signs were observed, euthanasia was carried out to detect the presence of tachyzoites in the peritoneal exudate. After the six weeks, the surviving animals were anesthetized and bled for anti-T. gondii antibody detection. The brain was also collected for histopathological analysis.

The organs collected after slaughter were fixed in 10% buffered formol for a week and blocked for histopathological studies. Two slides were stained with hematoxilin-eosin (HE). Immunohistochemistry (IHQ) was performed on the brain histological cuts and those with tissue cysts. The EnVision System Peroxidase (Dako Corp., Carpinteria, CA, USA) and the primary anti-T. gondii antibody (Dako Corp., Carpinteria, CA, USA) were used (ROSA, 1999).

Data regarding carcass weight and body temperature were analyzed by Anova (F distribution) and mean difference between experimental groups was performed by t test, at a = 5%.

RESULTS

Immunostimulating complex (iscoms) formation was confirmed by detection of cage-like structures using the electronic transmission microscope. Electrophoresis in polyacrilamide gel (SDS - 12%) showed that the main surface proteins, named P22 (SAG3), P23, P30 (SAG1), P35 and P43 (SAG3), were conserved in the iscoms.

One animal from G3 died on the 64^th day after infection (DAI) due to peritonitis by a perforating body. Three animals from G2 had clinical signs coinciding with the raise in temperature (40.9 to 41.0ºC) on the seventh and eighth day after the challenge (DAC). One animal from G4 showed hyperthermia (40.7ºC) on the sixth DAC without other clinical signs. The mean rectal temperatures remained within the standards for the species and age, although the statistical analysis showed significant differences (P<0.05) between G2, G3 (µ=39.436; µ=39.082, respectively) and G3, G4 (µ=39.082; µ=39.384, respectively) after challenge. Carcass mean weight, for all groups, did not show significant difference (P<0.05).

Table 1 shows the IFAT results. All the vaccinated animals (G1) showed an antigenic response after the first booster, while the animals which had received the antigenic particles without the Quil A adjuvant (G2) seroconverted only after the challenge. Animals in G3, immunized with RH sample tachyzoites, showed humoral response only after the second inoculation. Animals in G4 responded to the challenge with AS-28 sample oocysts and those in G5 remained negative throughout the experimental period.

The Western blotting test was performed using serum samples collected from animals in G1 (vaccinated with iscoms) after the second booster (56^th DAI) and after the challenge (86^th DAI) and serum samples from G4 animals (challenge group) collected after the challenge (86^th DAI).

The test showed the presence of 19 different protein bands after the second booster and 31 after the challenge in animals in G1. The proteins showing stronger reactions were those with molecular weight of 84, 75, 57, 31 kDa and those of 75, 63, 57, 31, 29, 27 and 25 kDa, respectively. A total of 22 protein bands were seen in G4 animals and stronger reactions were seen on those with molecular weight of 84, 61 and 31 kDa (Fig. 2).

The experimental inoculation of mice showed the presence of tachyzoites in the peritoneal exudate of one G2 animal inoculated with heart and two G4 animals inoculated with retina and heart. IFAT showed positive reaction (> 1:64) in one G3 animal inoculated with brain and in two G4 animals inoculated with tongue and retina. No tissue cysts were found in the brains in the histological analysis.

No cysts were seen in tissues collected from any of the animals. The IHQ carried out on the brain slides did not show neither T. gondii cysts, nor pseudocysts and tachyzoites formations.

DISCUSSION

Regarding all the clinical signs observed, animals in G1 remained healthy throughout the experimental period, confirming the protection induced by the iscoms for T. gondii infection. Three animals from the group immunized with the antigen (G2) and one from the non vaccinated group (G4) showed clinical signs including hypothermia and prostration after the challenge. Vidotto et al. (1987a,b) used 10⁴ oocysts of the same isolate to inoculate pregnant sows and reported clinical signs of toxoplasmosis on the fourth DAI, including abortion and fetal mummification. This antigenic variability within the same isolate may have occurred due to the number of successive inoculation in mice to maintain the AS-28, which could have altered its antigenic characteristics and consequently its pathogenicity (Frenkel, 1973). The loss of the ability to induce oocyst production in felines was another alteration observed by Frenkel et al. (1976), due successive inoculations in mice. Animals vaccinated with iscoms (G1) showed high levels of anti T. gondii antibodies (IgG) and, after the challenge, an increase of up to sixteenfold was observed in this titer in six out of nine animals in the group. Vaccination with T. gondii antigens without Quil A (G2) showed immunological weakening which emphasized the importance of this adjuvant. Lunden (1995) vaccinated adult sheep with T. gondii RH sample antigens associated to the iscoms and also obtained satisfactory results with good humoral and cellular immune response. Morein et al. (1987) reported that the antibody titers induced by the iscoms are 10 or more times superior to those induced by dead microorganisms or even inserted in micelles.

The proteins recognised by serum samples collected at different experimental phases had molecular weights of approximately 84, 75, 63, 57 and 31 kDa. Lundén (1995) used Western blotting in a similar experiment with sheep and found the presence of proteins with approximate molecular weights of 77, 63, 57, 42, 39, 37 34 kDa and specially those with 32 and 19 kDa, which showed a migration pattern similar to P30 and P22. The 52 and 29 kDa protein bands were also visualized after the challenge. Comparing the results of the present study to those reported by Lundén (1995), there is similarity in the proteins with molecular weight of 63, 57 and 31/30 kDa.

P30 (SAG3) and P22 (SAG 2) are present on the tachyzoite surface and are very important in the immune process (Boothroyd et al., 1998). P30 can induce a greater release of IFN-g and stimulate the T cytotoxic cells to an antigen-specific parasiticide effect, regardless of opsonization by antibodies or of Natural Killer cell activity (Khan et al., 1988). The presence of P30 in the vaccinated animals was essential for the stimulus of both the humoral and cell immune response.

P57 is one of the T. gondii excreted/secreted antigens (ESA) present in the organelles of the apical complex (micronemas, dense granules or roptrias) of the parasite and has been implicated in cell invasion, in the formation of the parasitophorous vacuole and its maintenance (Sam-Yellowe, 1996). Decoster et al. (1988) showed that these antigens are highly immunogenic in congenital or acquired human infections. In the present study, iscom vaccine stimulated P57 production, as shown in the Western blotting analysis.

The humoral immune response was more accentuated in the vaccinated and challenged animals and, although the cellular immune response was not measured, it can be inferred that the cell immunogenic stimulus was similar to the humoral, since the iscoms activate the CD4+ Th1 and CD8+ Tc cells (Barr, Graham, 1996).

Euthanasia was made 47 days after the challenge, at the chronic stage of infection, except for two animals from G2 that were undergoing the acute stage of infection. T. gondii re-isolation by bioassay was not frequent, but it was possible from four animals in the non-vaccinated group (G4), from one animal in the RH infected group (G3) and from one animal in G2. Vidotto et al. (1987a,b) re-isolated T. gondii from 18 types of tissue collected from six pregnant sows that had been inoculated with 10⁴ AS-28 oocysts, of which five were euthanasied during the acute phase of toxoplasmosis and only one at 46 DAI. The re-isolation occurred only from the lymphonode and pancreas in the latter. The same authors did not find the parasite in histological cuts. Dubey et al. (1994) vaccinated swine with 10⁵ live RH sample tachyzoites, via IM, and after the challenge with 10⁵ oocysts from a mixture of 10 isolates, T. gondii was re-isolated from three of the eight vaccinated animals. Pinckney et al. (1994) also could not totally prevent tissue cyst formation in piglets vaccinated with live ts-4 sample tachyzoites challenged with 8x10⁴ oocysts from the GT-1 isolate.

The histological examinations (HE and IHQ) did not show the presence of T. gondii cysts in any of the animals in the five experimental groups. Viotti et al. (1995) compared the immunofluorescence (IF), peroxidase anti peroxidase (PAP) and HE techniques for T. gondii diagnosis in histological cuts and bioassays. In the animals slaughtered during the acute phase of infection, the parasite was isolated in at least four out of the seven tissues processed for the bioassay. However in pigs slaughtered during the chronic phase the parasite was not isolated in any of the tissues studied. The PAP and IF techniques were efficacious for verifying the presence of T. gondii only in animals slaughtered during the acute phase.

When the techniques used in this studied are assessed to confirm tissue cyst formation, the bioassay was more efficacious than HE and IHQ. This fact may be explained by the greater quantity of tissue processed (50g) for the bioassay in comparison to the number of histological cuts examined (two cuts for HE staining and four prepared for IHQ) in each tissue collected. The bioassay has been the chosen technique in several vaccine and epidemiological studies for direct T. gondii diagnosis (Pinckney et al. 1994; Dubey et al. 1995; Mateus-Pinilla et al. 1999).

Rosa (1999) inoculated nine goats with 10⁵ oocysts from the same AS-28 isolate used in the present study and, after 56 days, at the chronic infection stage, the animals were euthanased. T. gondii cysts were not found in the histological cuts of 16 tissues stained neither by HE nor by IHQ, but all the goats showed the parasite in at least one of the tissues tested in the bioassay. In the view of the low re-isolation of T. gondii in swine tissue in comparison to the caprine species infected with the same isolate (AS-28) and the same parasite development stage (oocysts), it is supposed that tissue cyst formation is dependent, among other factors, on the animal species.

It was concluded that Toxoplasma vaccine iscoms are effective in the development of humoral immune response in pigs, but it is not possible to ensure, without further studies, that the immunized animals do not form tissue cysts after being challenged with T. gondii.

Recebido para publicação em 6 de outubro de 2002

Recebido para publicação, após modificações, em 23 de abril de 2003

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Publication Dates

• Publication in this collection
  12 Nov 2003
• Date of issue
  Aug 2003

History

• Accepted
  23 Apr 2003
• Received
  06 Oct 2002