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Excretory–secretory antigens: A suitable candidate for immunization against ocular toxoplasmosis in a murine model
Accepted Manuscript Title: Excretory-secretory antigens: a suitable candidate for immunization against ocular toxoplasmosis in a murine model Author: Kiumars Norouzpour Deilami Ahmad Daryani Ehsan Ahmadpour Mehdi Sharif Yousef Dadimoghaddam Shahabeddin Sarvi Ahad Alizadeh PII: DOI: Reference:
S0147-9571(14)00056-3 http://dx.doi.org/doi:10.1016/j.cimid.2014.10.003 CIMID 984
To appear in: Received date: Revised date: Accepted date:
10-6-2014 19-10-2014 20-10-2014
Please cite this article as: Deilami KN, Daryani A, Ahmadpour E, Sharif M, Dadimoghaddam Y, Sarvi S, Alizadeh A, Excretory-secretory antigens: a suitable candidate for immunization against ocular toxoplasmosis in a murine model, Comparative Immunology, Microbiology and Infectious Diseases (2014), http://dx.doi.org/10.1016/j.cimid.2014.10.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Highlights:
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We monitored the Toxoplasma distribution and parasite load determination using QPCR.
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We examined the ESAs of toxoplasma as a vaccine and evaluated parasite load in eye tissue in mice.
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ESAs in eye tissue of immunized mice compared to control mice has decreased parasite load.
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Excretory-secretory antigens: a suitable candidate for immunization against
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ocular toxoplasmosis in a murine model
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Kiumars Norouzpour Deilamia,b, Ahmad Daryania,c,*, Ehsan Ahmadpoura,c,*, Mehdi Sharif Yousef Dadimoghaddama,c, Shahabeddin Sarvi a,c, Ahad Alizadeh d
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Toxoplasmosis Research Center, Mazandaran University of Medical Sciences, Sari, Iran
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Department of Ophthalmology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran
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Department of Parasitology and Mycology, Sari Medical School, Mazandaran University of Medical Sciences, Sari, Iran
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Biostatistics Department, Mazandaran University of Medical Sciences, Sari, Iran
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Corresponding Author: Dr. Ahmad Daryani, Ehsan Ahmadpour,
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Toxoplasmosis Research Center, Mazandaran University of Medical Sciences, Sari Medical School, 18th Km of Khazar Abad Road, Sari, Iran
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Email address: [email protected], [email protected]
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Abstract
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Toxoplasmosis, responsible for ocular impairment, is caused by Toxoplasma gondii. We
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investigated the effect of Toxoplasma excretory-secretory antigens (ESA) on parasite load and
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distribution in the eye tissue of a murine model. Case and control groups were immunized with
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ESA and PBS, respectively. Two weeks after the second immunization, the mice were
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challenged intraperitoneally with virulent RH strain of Toxoplasma; eye tissue samples of both
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groups were collected daily (days 1, 2, 3, and the last day before death). Parasite load was
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determined using real-time quantitative PCR targeted at the B1 gene. Compared to the control
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group, infected mice that received ESA vaccine presented a considerable decrease in parasite
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load in the eye tissue, demonstrating the effect of ESA on parasite load and distribution.
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Diminution of parasite load in mouse eye tissue indicated that ESA might help control disease-
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related complications and could be a valuable immunization candidate against ocular
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toxoplasmosis.
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Keywords: Ocular toxoplasmosis, excretory-secretory antigens, vaccine, parasite load, Q-PCR
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1. Introduction
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Toxoplasmosis, caused by the intracellular protozoan parasite Toxoplasma gondii, is a major
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public health concern and the leading cause of disease in foetuses, new-borns, and immune-
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compromised patients [1, 2]. T. gondii mainly affects the central nervous system and the eyes.
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Ocular toxoplasmosis is one of the most important clinical manifestations of T. gondii infection
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and continues to be a poorly understood disease [3, 4]. Most cases of ocular toxoplasmosis occur
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through the congenital infection route (prenatally acquired). However, in some uncommon cases,
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the disease is caused by adult-acquired infections. The classic clinical symptoms in patients
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afflicted with ocular toxoplasmosis are pain, redness, floaters, blurred vision, photophobia, and
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blindness [1, 3, 4]. According to recent studies, ocular disease develops in 2%–20% of acquired
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toxoplasmosis cases and 70%–90% of toxoplasmosis cases caused by congenital infection.
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Higher rate of ocular toxoplasmosis may be related to host genetics and age, intensity and
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frequency of infection, manner of transmission, and, chiefly, strain and virulence of the parasite
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[5-9].
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Toxoplasmosis, a preventable cause of visual impairment and blindness, can be managed and
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cured successfully, if recognized early and treated appropriately [1, 4]. Development of a vaccine
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against toxoplasmosis is based on the vaccine’s ability to prevent harmful effects of the disease.
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Although Toxovax (S48 strain), a live vaccine, has been effective in preventing toxoplasmosis in
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sheep, it is unfortunately not suitable for human use. Thus, the vaccine against human
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toxoplasmosis remains an unsolved problem [10, 11].
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T. gondii expresses several antigens that have been identified as potential vaccine candidates.
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Moreover, these antigens play an important role in the pathogenesis of the microorganism, as
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well as in the parasite’s evasion of host immune responses. The excretory-secretory antigens
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(ESA) of the parasite that induce immunity in the hosts are suitable candidates for vaccination
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against T. gondii infection [12-15]. We previously evaluated the ESAs of T. gondii in mice
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immunization trials, which showed that compared to the control, the immunized mice survived
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for a longer period [16]. Based on animal studies, proliferating microorganisms are believed to
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be responsible for tissue destruction in retinochoroiditis. However, they are not normally
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associated with the immunological reaction to Toxoplasma antigens. One approach to
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toxoplasmosis vaccine development is the reduction of parasite number in the various tissues of
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the vaccinated host [2, 3, 13]. Hence, the present study was designed for the determination of T.
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gondii parasite load in the eye tissues of mice immunized with ESA of RH strain T. gondii using
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real-time quantitative polymerase chain reaction (QPCR).
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2. Material and Methods
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2.1. T. gondii strain
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The RH strain of T. gondii was used in the experiments. The tachyzoites were harvested from the
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peritoneal cavity of Swiss-Webster mice 3–4 days after intraperitoneal (i.p.) injection with 0.5 ml
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of parasite suspension prepared in sterile phosphate-buffered saline (PBS; pH 7.4) containing
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100 IU/ml penicillin and 100 µg/ml streptomycin. Peritoneal cells and debris were removed by
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washing with PBS and centrifugation at 800 × g for 10 min at 4°C. The number of parasites was
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counted using a haemocytometer under a light microscope (× 400 magnification) [16, 17].
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2.2. Preparation of excretory-secretory antigens
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ESAs were obtained according to a method previously described [16]. Briefly, T. gondii
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tachyzoites (ranging from 5 to 8 × 106 tachyzoites/ml) were incubated in 2 ml RPMI-1640
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containing 100 IU/ml penicillin and 100 µg/ml streptomycin at 37°C for 3 hours, with gentle
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shaking under sterile conditions. The tubes were then centrifuged at 1000 × g for 10 min and the
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supernatants were collected, pooled, and filtered through a 0.22-µm membrane filter (Millipore,
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Billerica, MA, USA). Protein concentration in the supernatants was determined using the
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Bradford method before storing them at −20°C [12, 16, 18-20].
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2.3. Mouse immunization and challenge experiments
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Inbred female C57BL/6 mice (age, 8–10 weeks), obtained from the Mazandaran University of
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Medical Sciences (Sari-Iran) animal house, were used for immunization and challenge
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experiments. Thirty-six female C57BL/6 mice were randomly divided into two groups (n = 18
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each), which were immunized with 40 µl of PBS emulsified with 40 µl of Freund's complete
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adjuvant (FCA) (negative control group) or 40 µl of ESA emulsified with an equal volume of
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FCA (case group). Immunization was performed subcutaneously in two doses at an interval of 2
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weeks. Subsequently, both mouse groups were challenged intraperitoneally with 1 × 104 live
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tachyzoites of RH strain to evaluate parasitaemia in eye tissues at days 1, 2, 3, and the last day
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before death (3 mice were sampled each day ) [16]. The project was reviewed and approved by
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the Ethics Committee of Mazandaran University of Medical Sciences.
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2.4. DNA extraction
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Total DNA was extracted from the eye tissue and the parasites by using the Tissue DNA
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Extraction Kit (Bioneer, Daejeon, Republic of Korea). DNA samples were dissolved in 200 µl
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Tris-EDTA buffer (TEB, pH 8.0).
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2.5. Real-time QPCR
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The highly conserved B1 gene of T. gondii, consisting of 35 copies of a 2214-base pair (bp)
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fragment was used to amplify a 451-bp fragment. The specific primers used for the amplification
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were the forward primer 5'-CTC CTT CGT CCG TCG TAA TAT C-3' and the reverse primer 5'-
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TGG TGT ACT GCG AAA ATG AAT C-3'. Real-time QPCR was performed in triplicate by
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using the SYBR Green Master Mix (Bioneer), 50 ng template DNA, 6 µl distilled water, and 10
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pmol of each primer in a final volume of 25 µl. Real-time QPCR was optimized under the
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following conditions: initial denaturation at 94°C for 3 min, followed by 40 cycles at 94°C for
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30 s (denaturation), 62°C for 40 s (annealing), and 72°C for 50 s (extension), followed by a final
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extension at 72°C for 5 min. Melting curve analysis was performed to verify the correct product
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size. The threshold cycle (CT) values determined on the basis of a standard curve obtained from
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the genome of RH strain tachyzoites serially diluted to specific concentrations (5 × 101–5 × 106
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copies/µl) were used to determine the parasite’s copy number. The results were expressed as T.
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gondii tachyzoite equivalents per mg of tissue. Real-time QPCR was analytically validated using
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the DNA of RH strain tachyzoites as positive control and peripheral blood DNA of uninfected
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mice as negative control.
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2.6. Statistical analysis
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The median number of tachyzoites per g per eye tissue sample collected from each group of mice
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at different times was calculated to check for statistical significance. Mann–Whitney U test and
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repeated measure ANOVA test were performed using the SPSS software (IBM, Armonk, NY,
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USA), and the values were considered to be significant when P < 0.05.
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3. Results
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In this survey, the B1 gene was used for T. gondii genome amplification. In order to assess the
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amplification efficiency and obtain the QPCR CT value, the prepared artificial samples
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containing various amounts of purified T. gondii DNA (0.05, 0.1, 1, 5, 50, 500, and 5000
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parasites/ml) and DNA from uninfected mouse eye tissue samples were amplified.
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Figure 1 shows the successful amplification of the 451-bp fragment of the B1 gene of T. gondii
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DNA. There was no amplification of DNA from uninfected mouse eye tissue samples. By using
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serial dilutions of T. gondii DNA to prepare standard curves, the linear equation and correlation
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coefficient were obtained as Y = −3.954x + 34.89 and R2 = 0.909, respectively.
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Mice were injected with T. gondii ESA emulsified with FCA to induce protective immunity
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against the disease. The control group was only injected with PBS mixed with FCA. All mice
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from case and control groups were euthanized, and the eye tissue samples were collected.
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Following DNA extraction, the parasite load in the eyes was carefully monitored by QPCR.
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Quantitative analysis of eye tissue samples collected on days 1, 2, and 3 post-inoculation (dpi)
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and before death allowed the detection and determination of the number of Toxoplasma DNA in
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all infected mice. The results obtained from the QPCR analyses of eye tissue samples evaluated
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for the presence of T. gondii (using CT) are shown in Table 1.
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Over the period of study, parasite burden-related outbursts were observed to occur in all the
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inoculated mice. All samples obtained from case and control groups were found to be positive
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for T. gondii.
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The results demonstrated that in the control group, the lowest CT (5 ± 3.68) or the highest copy
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number of T. gondii was seen in the eye tissue samples at day 2 dpi. In contrast, in the case
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group, the highest CT (25.17 ± 2.69) or the lowest copy number of T. gondii was observed at day
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2 dpi. It must be noted that the results indicated a significant association between the case and
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control groups (P = 0.0001). Furthermore, significant differences were observed at 2, 3, and the
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last dpi (P < 0.05). Accordingly, compared to the control group, infected mice that received the
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ESA solution displayed an increase of CT or a drastic decrease in parasite load or circulating
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parasite DNA levels. As shown in Figure 2, administration of the ESA prevented parasite
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dissemination in mouse eye tissue.
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4. Discussion
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This study demonstrates that T. gondii ESA have a superior effect in reducing parasite load in the
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eye tissue and provide significant protection against tachyzoite distribution following
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intraperitoneal injection to abrogate ocular toxoplasmosis. Among the different antigens of
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Toxoplasma, it has been demonstrated that ESA are highly immunogenic and stimulate both
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humoral and cellular immune responses [12, 16]. These antigens are released by secretory
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organelles like micronemes, rhoptries, and dense granules [14, 21, 22]. Numerous experimental
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studies have been conducted on the effect of ESA in animal models; however, none has focused
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on the tissue tropism aspect of parasites. The tachyzoite kinetics in the host body, specifically the
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eye, show that T. gondii causes severe complications such as chorioretinitis [23]. Therefore, in
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addition to evaluating the immunogenicity of the vaccine, determination of movement pattern
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and tissue parasite load after vaccination is also required. Ocular toxoplasmosis still poses
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several questions that confuse researchers and ophthalmologists. Besides the genetic background
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of the host and the parasite genotypes (type I, II, and III strains) that have been known to damage
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the ocular tissues, the host immune response is likely to play an important role in retarding
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disease progression, and, possibly, in successful response to therapy [24-26]. Several studies
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have been conducted on quantification and determination of parasite load in different tissues
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over the past decade. However, there have been few experimental reports concerning acquired
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ocular toxoplasmosis in animal models [31-33].
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Many factors may play an important role in induction of immune response against T. gondii
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infection. In addition to the mouse and T. gondii strains, the choice of adjuvants plays an
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important role in determining the efficacy of immunization.
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Prior studies in this field most commonly include experiments that have used Balb/c mice and
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the RH strain of Toxoplasma. Based on our previous research on T. gondii ESA and their use in
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immunization [16, 34, 35], we have, in this study, monitored and quantified for the first time, the
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distribution and parasite load of T. gondii RH strain in the eye tissue of mice immunized with
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ESA. The aim of the study was to evaluate the parasite load after immunization by ESA, in
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throughout the eye tissue, not only at a part of eye such as Uvea. On the other hand, since T.
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gondii RH pathogen strain with high virulence is a proper strain for evaluation of parasite load in
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different tissues (34) including eye; therefore in this study we used RH strain. By the way, since
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ESA has been decreased parasite load of a pathogen strain, RH, probably it can decrease a low
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pathogen strain such as Me49. Of course, we will research in this field in the future. In this study,
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the ESA were supplemented with a strong adjuvant (Freund’s adjuvant) in order to enhance
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antigen-specific immune responses. It has been previously demonstrated that protection against
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T. gondii in mice is related to Th1 cell-mediated immunity, as well as to the secretion of IFN and
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induction of specific B-cell responses. Although aluminium-derived adjuvants (alum) are often
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used in clinical trials for human and animal vaccines, its stimulation mechanism on the immune
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system and its effect on cell-mediated immunity remain unknown. A number of immunization
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studies have used Freund’s adjuvant in animal experimentation. Gatkowska et al. have
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demonstrated a high protective efficacy of recombinant antigens supplemented with Freund’s
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adjuvants (36). Wang et al. showed that immunization with ESA emulsified in Freund's adjuvant
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induced humoral (antibody-mediated), as well as cellular immune responses (T cell-mediated)
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and produced a highly effective and significant protection against T. gondii infection (37, 38).
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For the challenge experiments, we injected the mice with 1 × 104 live tachyzoites. The mice from
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the control group survived for 4 days, while mice immunized with ESA survived for more than
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10 days. Since the survival time for the case and control groups was not the same and the
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comparison of parasite load in case and control groups was to be done daily, we presented the
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data for days 1, 2, 3, and the last day before death.
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The mechanism by which the tachyzoite-infected cells adhere to the tissues is unknown. Unno et
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al. [2013] reported that Toxoplasma infection alters the expression levels of host adhesion
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molecules on the cell surface (39). Such an alteration of the adhesion molecules on the surface of
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peripheral blood mononuclear cells (PBMCs) might facilitate T. gondii dissemination into the
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peripheral organs of the host [27]. As demonstrated by Norose et al. (2003), IFN-γ regulates T.
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gondii distribution and load in the eye tissue [28].
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In ocular toxoplasmosis, percolation of the tachyzoites through the blood–ocular barrier is an
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important mechanism during the initial invasion of the eye tissue [29]. It has also been suggested
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that tachyzoites may be transmitted from the brain through the optic nerve [23]. Furthermore,
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Furtado et al. (2012) demonstrated that the dendritic cells act as a potential taxi for tachyzoites
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and that transmigration of these cells is increased following Toxoplasma infection, probably
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owing to locally produced chemokines [30].
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The dissemination of Toxoplasma has been previously examined by conventional methods such
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as Giemsa staining, subinoculation of various tissues into mice, and tissue culture of serial
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dilutions of organ homogenates [18]. However, techniques that involve calculating the number of
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infected and non-infected PBMCs in tissue sections are cumbersome, inaccurate, and inefficient
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[39].
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In our study, we have used the real-time QPCR technique and the B1 gene for the quantification
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of tachyzoites in the eye tissue samples of experimentally infected mice. Real-time QPCR assay
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is known to be used for the detection, quantification, and estimation of parasite load in different
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tissues. QPCR is reliable, accurate, and can detect as less as 0.1 pg parasite DNA [32]. After the
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introduction of PCR-based molecular methods, especially quantitative PCR, there have been a
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few reports on the analysis of the distribution of Toxoplasma in experimental murine infections
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by using QPCR. Previous studies have shown that QPCR is a sensitive, fast, and specific method
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for parasite load assessment and for monitoring T. gondii in different tissues. This approach
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could also be a proper alternative for routine screening in clinical laboratories [31-33, 40].
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In this study, we have demonstrated basic parasitological parameters such as the time of
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appearance and the number of T. gondii during the natural course of infection in both case and
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control groups. It appears that the natural tropism of the parasite to different tissues is time
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dependent; after filtration and elimination of parasites from the spleen, followed by its filtration
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from the kidney, the parasite load (T. gondii tachyzoites or DNA) was seen to reduce. Our results
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indicated that on the second day after infection, the natural tropism of the parasite (presence and
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proliferation of T. gondii) towards eye tissue was higher than that at other times. The protection
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afforded to case group mice by subcutaneous injection of ESA significantly reduced the parasite
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load when compared to that observed in the non-immunized mice from the control group (P =
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0.0001). The large amounts of parasite DNA observed in the eye tissue after the experimental i.p.
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infection can be explained by the known neurotropism and ocular tropism of Toxoplasma RH
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strain.
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As mentioned in previous vaccination studies, there is a paucity of research regarding the effects
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of vaccination on parasite burden, particularly in the eye tissue [11, 13, 41, 42]. Evaluation and
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assessment of the Toxoplasma vaccine, which prevents ocular or cerebral localization of the
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parasite in animal models, should be helpful in the control of toxoplasmosis. It is interesting that
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administration of ESA in eye tissue, where the immune system does not have access, helps
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decrease the parasite load. In fact, ESA were seen to stimulate the cellular and humoral immune
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response in mice, and after the challenge experiment, were found to inhibit parasite proliferation,
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thereby resulting in lower numbers of Toxoplasma tachyzoites being distributed in tissues.
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5. Conclusion
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In summary, we have demonstrated the use of Toxoplasma ESA as a vaccine against
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toxoplasmosis, which induced a marked decrease in parasite load and prevented tachyzoite
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distribution in the eye tissue. Therefore, ESA could be valuable immunization candidates against
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ocular toxoplasmosis.
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Conflict of interest
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The authors declared no conflicts of interest.
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Acknowledgments
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This study received financial support (grant no. 90-31) from Deputy of Research, Mazandaran
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University of Medical Sciences, Sari, Iran.
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Carruthers VB, Giddings OK, Sibley LD. Secretion of micronemal proteins is associated
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Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities
Khan A, Jordan C, Muccioli C, Vallochi AL, Rizzo LV, Belfort Jr R, et al. Genetic
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divergence of Toxoplasma gondii strains associated with ocular toxoplasmosis, Brazil.
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Emerg Infect Dis 2006;12:942.
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Unno A, Suzuki K, Xuan X, Nishikawa Y, Kitoh K, Takashima Y. Dissemination of
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extracellular and intracellular Toxoplasma gondii tachyzoites in the blood flow. Parasitol
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Int 2008;57:515-8.
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Norose K, Mun H-S, Aosai F, Chen M, Piao L-X, Kobayashi M, et al. IFN-γ Regulated
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Toxoplasma gondii Distribution and Load in the Murine Eye. Invest Ophth Vis Sci
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Toxoplasma gondii through the blood - brain barrier. J Neuroimmunol 2011;232:119-30.
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Lachenmaier SM, Deli MA, Meissner M, Liesenfeld O. Intracellular transport of
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Furtado JM, Bharadwaj AS, Ashander LM, Olivas A, Smith JR. Migration of Toxoplasma gondii Infected Dendritic Cells across Human Retinal Vascular Endothelium. Invest Ophth
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Vis Sci 2012;53:6856-62. 31.
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Aigner CP, Silva AVd, Sandrini F, Osorio PdS, Poiares L, Largura A. Real-time PCRbased quantification of Toxoplasma gondii in tissue samples of serologically positive
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outdoor chickens. Mem Inst Oswaldo Cruz 2010;105:935-7. 32.
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Lin M-H, Chen T-C, Kuo T-T, Tseng C-C, Tseng C-P. Real-Time PCR for Quantitative Detection of Toxoplasma gondii. J Clin Microbiol 2000;38:4121-5.
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33. Piña-Vázquez C, Saavedra R, Herion P. A quantitative competitive PCR method to
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determine the parasite load in the brain of Toxoplasma gondii-infected mice. Parasitol Int
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2008;57:347-53.
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34. Daryani A, Sharif M, Dadimoghaddam Y, Hashemi Souteh M. B, Ahmadpour E, Khalilian
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A, et al. Determination of parasitic load in different tissues of murine toxoplasmosis after
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immunization by excretory–secretory antigens using Real time QPCR. Exp Parasitol 2014;
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143:55-59.
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35. Daryani A, Sharif M, Kalani H, Rafiei A, Kalani F, Ahmadpour E. Electrophoretic Patterns
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of Toxoplasma gondii Excreted/Secreted Antigens and Their Role in Induction of the
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Humoral Immune Response. JJM 2014;7:E9225.
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36. Gatkowska J, Gasior A, Kur J, Dlugonska H. Toxoplasma gondii: Chimeric Dr fimbriae as a recombinant vaccine against toxoplasmosis. Exp Parasitol 2008;118:266-270.
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37. Wang Y, Wang M, Wang G, Pang A, Fu B, Yin H, et al. Increased survival time in mice
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vaccinated with a branched lysine multiple antigenic peptide containing B-and T-cell
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epitopes from T. gondii antigens. Vaccine 2011; 29:8619-8623.
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38. Wang Y, Zhang D, Wang G, Yin H, Wang M. Immunization with excreted–secreted antigens
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reduces tissue cyst formation in pigs. Parasitol Res 2013;112:3835-3842.
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Unno A, Kachi S, Batanova TA, Ohno T, Elhawary N, Kitoh K, et al. Toxoplasma gondii
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tachyzoite-infected peripheral blood mononuclear cells are enriched in mouse lungs and
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liver. Exp Parasitol 2013;134:160-4.
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40.
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Kompalic-Cristo A, Frotta C, Suárez-Mutis M, Fernandes O, Britto C. Evaluation of a realtime PCR assay based on the repetitive B1 gene for the detection of Toxoplasma gondii in
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human peripheral blood. Parasitol Res 2007;101:619-25. Waldeland H, Frenkel JK. Live and killed vaccines against toxoplasmosis in mice. J Parasitol 1983:60-5.
374 375
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42.
Zhang M, Zhao L, Song J, Li Y, Zhao Q, He S, et al. DNA vaccine encoding the
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Toxoplasma gondii bradyzoite-specific surface antigens SAG2CDX protect BALB/c mice
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against type II parasite infection. Vaccine 2013;31:4536-40.
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Figure 1: QPCR method detection and quantification of the tachyzoite copy number (parasite
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load). A: QPCR standard curve, ● Standard samples with known amounts tachyzoites number, ×
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Unknown samples. B: The QPCR amplification products from section A were analyzed by
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agarose gel electrophoresis (1.5%). M: DNA molecular weight marker (100bp), P: positive
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control (50 pg of tachyzoite DNA), Lane 1, 2: unknown samples from eye tissue, N: negative
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control.
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14000000 12000000
8000000 Case
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Control 2171406 Case
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Figure 2: Comparison of estimated number of tachyzoites in eye tissue between case and control
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S0147-9571(14)00056-3 http://dx.doi.org/doi:10.1016/j.cimid.2014.10.003 CIMID 984
To appear in: Received date: Revised date: Accepted date:
10-6-2014 19-10-2014 20-10-2014
Please cite this article as: Deilami KN, Daryani A, Ahmadpour E, Sharif M, Dadimoghaddam Y, Sarvi S, Alizadeh A, Excretory-secretory antigens: a suitable candidate for immunization against ocular toxoplasmosis in a murine model, Comparative Immunology, Microbiology and Infectious Diseases (2014), http://dx.doi.org/10.1016/j.cimid.2014.10.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Highlights:
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We monitored the Toxoplasma distribution and parasite load determination using QPCR.
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We examined the ESAs of toxoplasma as a vaccine and evaluated parasite load in eye tissue in mice.
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ESAs in eye tissue of immunized mice compared to control mice has decreased parasite load.
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Excretory-secretory antigens: a suitable candidate for immunization against
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ocular toxoplasmosis in a murine model
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Kiumars Norouzpour Deilamia,b, Ahmad Daryania,c,*, Ehsan Ahmadpoura,c,*, Mehdi Sharif Yousef Dadimoghaddama,c, Shahabeddin Sarvi a,c, Ahad Alizadeh d
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Toxoplasmosis Research Center, Mazandaran University of Medical Sciences, Sari, Iran
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Department of Ophthalmology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran
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c
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Department of Parasitology and Mycology, Sari Medical School, Mazandaran University of Medical Sciences, Sari, Iran
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Biostatistics Department, Mazandaran University of Medical Sciences, Sari, Iran
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Corresponding Author: Dr. Ahmad Daryani, Ehsan Ahmadpour,
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Toxoplasmosis Research Center, Mazandaran University of Medical Sciences, Sari Medical School, 18th Km of Khazar Abad Road, Sari, Iran
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Email address: [email protected], [email protected]
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Abstract
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Toxoplasmosis, responsible for ocular impairment, is caused by Toxoplasma gondii. We
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investigated the effect of Toxoplasma excretory-secretory antigens (ESA) on parasite load and
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distribution in the eye tissue of a murine model. Case and control groups were immunized with
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ESA and PBS, respectively. Two weeks after the second immunization, the mice were
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challenged intraperitoneally with virulent RH strain of Toxoplasma; eye tissue samples of both
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groups were collected daily (days 1, 2, 3, and the last day before death). Parasite load was
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determined using real-time quantitative PCR targeted at the B1 gene. Compared to the control
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group, infected mice that received ESA vaccine presented a considerable decrease in parasite
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load in the eye tissue, demonstrating the effect of ESA on parasite load and distribution.
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Diminution of parasite load in mouse eye tissue indicated that ESA might help control disease-
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related complications and could be a valuable immunization candidate against ocular
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toxoplasmosis.
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Keywords: Ocular toxoplasmosis, excretory-secretory antigens, vaccine, parasite load, Q-PCR
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1. Introduction
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Toxoplasmosis, caused by the intracellular protozoan parasite Toxoplasma gondii, is a major
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public health concern and the leading cause of disease in foetuses, new-borns, and immune-
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compromised patients [1, 2]. T. gondii mainly affects the central nervous system and the eyes.
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Ocular toxoplasmosis is one of the most important clinical manifestations of T. gondii infection
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and continues to be a poorly understood disease [3, 4]. Most cases of ocular toxoplasmosis occur
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through the congenital infection route (prenatally acquired). However, in some uncommon cases,
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the disease is caused by adult-acquired infections. The classic clinical symptoms in patients
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afflicted with ocular toxoplasmosis are pain, redness, floaters, blurred vision, photophobia, and
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blindness [1, 3, 4]. According to recent studies, ocular disease develops in 2%–20% of acquired
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toxoplasmosis cases and 70%–90% of toxoplasmosis cases caused by congenital infection.
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Higher rate of ocular toxoplasmosis may be related to host genetics and age, intensity and
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frequency of infection, manner of transmission, and, chiefly, strain and virulence of the parasite
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[5-9].
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Toxoplasmosis, a preventable cause of visual impairment and blindness, can be managed and
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cured successfully, if recognized early and treated appropriately [1, 4]. Development of a vaccine
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against toxoplasmosis is based on the vaccine’s ability to prevent harmful effects of the disease.
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Although Toxovax (S48 strain), a live vaccine, has been effective in preventing toxoplasmosis in
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sheep, it is unfortunately not suitable for human use. Thus, the vaccine against human
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toxoplasmosis remains an unsolved problem [10, 11].
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T. gondii expresses several antigens that have been identified as potential vaccine candidates.
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Moreover, these antigens play an important role in the pathogenesis of the microorganism, as
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well as in the parasite’s evasion of host immune responses. The excretory-secretory antigens
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(ESA) of the parasite that induce immunity in the hosts are suitable candidates for vaccination
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against T. gondii infection [12-15]. We previously evaluated the ESAs of T. gondii in mice
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immunization trials, which showed that compared to the control, the immunized mice survived
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for a longer period [16]. Based on animal studies, proliferating microorganisms are believed to
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be responsible for tissue destruction in retinochoroiditis. However, they are not normally
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associated with the immunological reaction to Toxoplasma antigens. One approach to
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toxoplasmosis vaccine development is the reduction of parasite number in the various tissues of
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the vaccinated host [2, 3, 13]. Hence, the present study was designed for the determination of T.
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gondii parasite load in the eye tissues of mice immunized with ESA of RH strain T. gondii using
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real-time quantitative polymerase chain reaction (QPCR).
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2. Material and Methods
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2.1. T. gondii strain
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The RH strain of T. gondii was used in the experiments. The tachyzoites were harvested from the
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peritoneal cavity of Swiss-Webster mice 3–4 days after intraperitoneal (i.p.) injection with 0.5 ml
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of parasite suspension prepared in sterile phosphate-buffered saline (PBS; pH 7.4) containing
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100 IU/ml penicillin and 100 µg/ml streptomycin. Peritoneal cells and debris were removed by
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washing with PBS and centrifugation at 800 × g for 10 min at 4°C. The number of parasites was
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counted using a haemocytometer under a light microscope (× 400 magnification) [16, 17].
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2.2. Preparation of excretory-secretory antigens
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ESAs were obtained according to a method previously described [16]. Briefly, T. gondii
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tachyzoites (ranging from 5 to 8 × 106 tachyzoites/ml) were incubated in 2 ml RPMI-1640
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containing 100 IU/ml penicillin and 100 µg/ml streptomycin at 37°C for 3 hours, with gentle
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shaking under sterile conditions. The tubes were then centrifuged at 1000 × g for 10 min and the
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supernatants were collected, pooled, and filtered through a 0.22-µm membrane filter (Millipore,
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Billerica, MA, USA). Protein concentration in the supernatants was determined using the
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Bradford method before storing them at −20°C [12, 16, 18-20].
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2.3. Mouse immunization and challenge experiments
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Inbred female C57BL/6 mice (age, 8–10 weeks), obtained from the Mazandaran University of
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Medical Sciences (Sari-Iran) animal house, were used for immunization and challenge
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experiments. Thirty-six female C57BL/6 mice were randomly divided into two groups (n = 18
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each), which were immunized with 40 µl of PBS emulsified with 40 µl of Freund's complete
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adjuvant (FCA) (negative control group) or 40 µl of ESA emulsified with an equal volume of
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FCA (case group). Immunization was performed subcutaneously in two doses at an interval of 2
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weeks. Subsequently, both mouse groups were challenged intraperitoneally with 1 × 104 live
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tachyzoites of RH strain to evaluate parasitaemia in eye tissues at days 1, 2, 3, and the last day
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before death (3 mice were sampled each day ) [16]. The project was reviewed and approved by
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the Ethics Committee of Mazandaran University of Medical Sciences.
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2.4. DNA extraction
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Total DNA was extracted from the eye tissue and the parasites by using the Tissue DNA
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Extraction Kit (Bioneer, Daejeon, Republic of Korea). DNA samples were dissolved in 200 µl
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Tris-EDTA buffer (TEB, pH 8.0).
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2.5. Real-time QPCR
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The highly conserved B1 gene of T. gondii, consisting of 35 copies of a 2214-base pair (bp)
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fragment was used to amplify a 451-bp fragment. The specific primers used for the amplification
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were the forward primer 5'-CTC CTT CGT CCG TCG TAA TAT C-3' and the reverse primer 5'-
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TGG TGT ACT GCG AAA ATG AAT C-3'. Real-time QPCR was performed in triplicate by
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using the SYBR Green Master Mix (Bioneer), 50 ng template DNA, 6 µl distilled water, and 10
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pmol of each primer in a final volume of 25 µl. Real-time QPCR was optimized under the
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following conditions: initial denaturation at 94°C for 3 min, followed by 40 cycles at 94°C for
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30 s (denaturation), 62°C for 40 s (annealing), and 72°C for 50 s (extension), followed by a final
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extension at 72°C for 5 min. Melting curve analysis was performed to verify the correct product
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size. The threshold cycle (CT) values determined on the basis of a standard curve obtained from
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the genome of RH strain tachyzoites serially diluted to specific concentrations (5 × 101–5 × 106
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copies/µl) were used to determine the parasite’s copy number. The results were expressed as T.
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gondii tachyzoite equivalents per mg of tissue. Real-time QPCR was analytically validated using
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the DNA of RH strain tachyzoites as positive control and peripheral blood DNA of uninfected
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mice as negative control.
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2.6. Statistical analysis
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The median number of tachyzoites per g per eye tissue sample collected from each group of mice
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at different times was calculated to check for statistical significance. Mann–Whitney U test and
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repeated measure ANOVA test were performed using the SPSS software (IBM, Armonk, NY,
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USA), and the values were considered to be significant when P < 0.05.
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3. Results
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In this survey, the B1 gene was used for T. gondii genome amplification. In order to assess the
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amplification efficiency and obtain the QPCR CT value, the prepared artificial samples
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containing various amounts of purified T. gondii DNA (0.05, 0.1, 1, 5, 50, 500, and 5000
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parasites/ml) and DNA from uninfected mouse eye tissue samples were amplified.
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Figure 1 shows the successful amplification of the 451-bp fragment of the B1 gene of T. gondii
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DNA. There was no amplification of DNA from uninfected mouse eye tissue samples. By using
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serial dilutions of T. gondii DNA to prepare standard curves, the linear equation and correlation
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coefficient were obtained as Y = −3.954x + 34.89 and R2 = 0.909, respectively.
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Mice were injected with T. gondii ESA emulsified with FCA to induce protective immunity
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against the disease. The control group was only injected with PBS mixed with FCA. All mice
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from case and control groups were euthanized, and the eye tissue samples were collected.
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Following DNA extraction, the parasite load in the eyes was carefully monitored by QPCR.
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Quantitative analysis of eye tissue samples collected on days 1, 2, and 3 post-inoculation (dpi)
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and before death allowed the detection and determination of the number of Toxoplasma DNA in
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all infected mice. The results obtained from the QPCR analyses of eye tissue samples evaluated
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for the presence of T. gondii (using CT) are shown in Table 1.
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Over the period of study, parasite burden-related outbursts were observed to occur in all the
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inoculated mice. All samples obtained from case and control groups were found to be positive
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for T. gondii.
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The results demonstrated that in the control group, the lowest CT (5 ± 3.68) or the highest copy
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number of T. gondii was seen in the eye tissue samples at day 2 dpi. In contrast, in the case
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group, the highest CT (25.17 ± 2.69) or the lowest copy number of T. gondii was observed at day
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2 dpi. It must be noted that the results indicated a significant association between the case and
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control groups (P = 0.0001). Furthermore, significant differences were observed at 2, 3, and the
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last dpi (P < 0.05). Accordingly, compared to the control group, infected mice that received the
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ESA solution displayed an increase of CT or a drastic decrease in parasite load or circulating
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parasite DNA levels. As shown in Figure 2, administration of the ESA prevented parasite
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dissemination in mouse eye tissue.
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4. Discussion
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This study demonstrates that T. gondii ESA have a superior effect in reducing parasite load in the
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eye tissue and provide significant protection against tachyzoite distribution following
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intraperitoneal injection to abrogate ocular toxoplasmosis. Among the different antigens of
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Toxoplasma, it has been demonstrated that ESA are highly immunogenic and stimulate both
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humoral and cellular immune responses [12, 16]. These antigens are released by secretory
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organelles like micronemes, rhoptries, and dense granules [14, 21, 22]. Numerous experimental
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studies have been conducted on the effect of ESA in animal models; however, none has focused
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on the tissue tropism aspect of parasites. The tachyzoite kinetics in the host body, specifically the
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eye, show that T. gondii causes severe complications such as chorioretinitis [23]. Therefore, in
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addition to evaluating the immunogenicity of the vaccine, determination of movement pattern
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and tissue parasite load after vaccination is also required. Ocular toxoplasmosis still poses
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several questions that confuse researchers and ophthalmologists. Besides the genetic background
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of the host and the parasite genotypes (type I, II, and III strains) that have been known to damage
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the ocular tissues, the host immune response is likely to play an important role in retarding
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disease progression, and, possibly, in successful response to therapy [24-26]. Several studies
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have been conducted on quantification and determination of parasite load in different tissues
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over the past decade. However, there have been few experimental reports concerning acquired
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ocular toxoplasmosis in animal models [31-33].
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Many factors may play an important role in induction of immune response against T. gondii
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infection. In addition to the mouse and T. gondii strains, the choice of adjuvants plays an
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important role in determining the efficacy of immunization.
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Prior studies in this field most commonly include experiments that have used Balb/c mice and
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the RH strain of Toxoplasma. Based on our previous research on T. gondii ESA and their use in
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immunization [16, 34, 35], we have, in this study, monitored and quantified for the first time, the
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distribution and parasite load of T. gondii RH strain in the eye tissue of mice immunized with
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ESA. The aim of the study was to evaluate the parasite load after immunization by ESA, in
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throughout the eye tissue, not only at a part of eye such as Uvea. On the other hand, since T.
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gondii RH pathogen strain with high virulence is a proper strain for evaluation of parasite load in
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different tissues (34) including eye; therefore in this study we used RH strain. By the way, since
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ESA has been decreased parasite load of a pathogen strain, RH, probably it can decrease a low
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pathogen strain such as Me49. Of course, we will research in this field in the future. In this study,
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the ESA were supplemented with a strong adjuvant (Freund’s adjuvant) in order to enhance
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antigen-specific immune responses. It has been previously demonstrated that protection against
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T. gondii in mice is related to Th1 cell-mediated immunity, as well as to the secretion of IFN and
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induction of specific B-cell responses. Although aluminium-derived adjuvants (alum) are often
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used in clinical trials for human and animal vaccines, its stimulation mechanism on the immune
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system and its effect on cell-mediated immunity remain unknown. A number of immunization
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studies have used Freund’s adjuvant in animal experimentation. Gatkowska et al. have
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demonstrated a high protective efficacy of recombinant antigens supplemented with Freund’s
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adjuvants (36). Wang et al. showed that immunization with ESA emulsified in Freund's adjuvant
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induced humoral (antibody-mediated), as well as cellular immune responses (T cell-mediated)
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and produced a highly effective and significant protection against T. gondii infection (37, 38).
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For the challenge experiments, we injected the mice with 1 × 104 live tachyzoites. The mice from
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the control group survived for 4 days, while mice immunized with ESA survived for more than
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10 days. Since the survival time for the case and control groups was not the same and the
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comparison of parasite load in case and control groups was to be done daily, we presented the
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data for days 1, 2, 3, and the last day before death.
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The mechanism by which the tachyzoite-infected cells adhere to the tissues is unknown. Unno et
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al. [2013] reported that Toxoplasma infection alters the expression levels of host adhesion
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molecules on the cell surface (39). Such an alteration of the adhesion molecules on the surface of
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peripheral blood mononuclear cells (PBMCs) might facilitate T. gondii dissemination into the
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peripheral organs of the host [27]. As demonstrated by Norose et al. (2003), IFN-γ regulates T.
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gondii distribution and load in the eye tissue [28].
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In ocular toxoplasmosis, percolation of the tachyzoites through the blood–ocular barrier is an
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important mechanism during the initial invasion of the eye tissue [29]. It has also been suggested
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that tachyzoites may be transmitted from the brain through the optic nerve [23]. Furthermore,
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Furtado et al. (2012) demonstrated that the dendritic cells act as a potential taxi for tachyzoites
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and that transmigration of these cells is increased following Toxoplasma infection, probably
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owing to locally produced chemokines [30].
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The dissemination of Toxoplasma has been previously examined by conventional methods such
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as Giemsa staining, subinoculation of various tissues into mice, and tissue culture of serial
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dilutions of organ homogenates [18]. However, techniques that involve calculating the number of
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infected and non-infected PBMCs in tissue sections are cumbersome, inaccurate, and inefficient
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[39].
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In our study, we have used the real-time QPCR technique and the B1 gene for the quantification
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of tachyzoites in the eye tissue samples of experimentally infected mice. Real-time QPCR assay
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is known to be used for the detection, quantification, and estimation of parasite load in different
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tissues. QPCR is reliable, accurate, and can detect as less as 0.1 pg parasite DNA [32]. After the
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introduction of PCR-based molecular methods, especially quantitative PCR, there have been a
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few reports on the analysis of the distribution of Toxoplasma in experimental murine infections
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by using QPCR. Previous studies have shown that QPCR is a sensitive, fast, and specific method
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for parasite load assessment and for monitoring T. gondii in different tissues. This approach
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could also be a proper alternative for routine screening in clinical laboratories [31-33, 40].
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In this study, we have demonstrated basic parasitological parameters such as the time of
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appearance and the number of T. gondii during the natural course of infection in both case and
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control groups. It appears that the natural tropism of the parasite to different tissues is time
248
dependent; after filtration and elimination of parasites from the spleen, followed by its filtration
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from the kidney, the parasite load (T. gondii tachyzoites or DNA) was seen to reduce. Our results
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indicated that on the second day after infection, the natural tropism of the parasite (presence and
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proliferation of T. gondii) towards eye tissue was higher than that at other times. The protection
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afforded to case group mice by subcutaneous injection of ESA significantly reduced the parasite
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load when compared to that observed in the non-immunized mice from the control group (P =
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0.0001). The large amounts of parasite DNA observed in the eye tissue after the experimental i.p.
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infection can be explained by the known neurotropism and ocular tropism of Toxoplasma RH
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strain.
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As mentioned in previous vaccination studies, there is a paucity of research regarding the effects
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of vaccination on parasite burden, particularly in the eye tissue [11, 13, 41, 42]. Evaluation and
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assessment of the Toxoplasma vaccine, which prevents ocular or cerebral localization of the
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parasite in animal models, should be helpful in the control of toxoplasmosis. It is interesting that
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administration of ESA in eye tissue, where the immune system does not have access, helps
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decrease the parasite load. In fact, ESA were seen to stimulate the cellular and humoral immune
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response in mice, and after the challenge experiment, were found to inhibit parasite proliferation,
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thereby resulting in lower numbers of Toxoplasma tachyzoites being distributed in tissues.
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5. Conclusion
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In summary, we have demonstrated the use of Toxoplasma ESA as a vaccine against
267
toxoplasmosis, which induced a marked decrease in parasite load and prevented tachyzoite
268
distribution in the eye tissue. Therefore, ESA could be valuable immunization candidates against
269
ocular toxoplasmosis.
270
Conflict of interest
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The authors declared no conflicts of interest.
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Acknowledgments
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This study received financial support (grant no. 90-31) from Deputy of Research, Mazandaran
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University of Medical Sciences, Sari, Iran.
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Figure 1: QPCR method detection and quantification of the tachyzoite copy number (parasite
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load). A: QPCR standard curve, ● Standard samples with known amounts tachyzoites number, ×
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Unknown samples. B: The QPCR amplification products from section A were analyzed by
383
agarose gel electrophoresis (1.5%). M: DNA molecular weight marker (100bp), P: positive
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control (50 pg of tachyzoite DNA), Lane 1, 2: unknown samples from eye tissue, N: negative
385
control.
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Figure 2: Comparison of estimated number of tachyzoites in eye tissue between case and control
390
groups.
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