Amoebicidal activity of essential oils and essential oil-based microemulsions of Aloysia citrodora Ortega ex Pers., Cymbopogon winterianus Jowitt ex Bor, and Ocimum gratissimum L. against Acanthamoeba polyphaga trophozoites.

AIMS: Evaluate the in vitro efficacy of the essential oils derived from Aloysia citrodora (Verbenaceae), Cymbopogon winterianus (Poaceae), and Ocimum gratissimum (Lamiaceae) against Acanthamoeba polyphaga trophozoites. Additionally, microemulsions formulated with these essential oils, along with their major components, were analyzed. METHODS AND RESULTS: The prepared microemulsions were characterized using polarized light microscopy and rheological techniques. The amoebicidal activity was determined by measuring the inhibitory concentration (IC50). Flow cytometry was employed to detect membrane damage and alterations in trophozoites size. The results revealed transparent and thermodynamically stable microemulsions. The essential oil from O. gratissimum exhibited a lower IC50, with values of 280.66 and 47.28 µg ml-1 after 24 and 48 h, respectively. When microemulsions containing essential oils were tested, the IC50 values exhibited a reduction of over 80% after 24 h. Particularly, eugenol, a constituent of the O. gratissimum essential oil, displayed higher amoebicidal activity. The essential oils also caused damage to the cell membrane, resulting in the subsequent death of the trophozoites. CONCLUSIONS: The EOs of A. citrodora, C. winterianus, and O. gratissimum and their microemulsions showed antiparasitic effect against A. polyphaga trophozoites, representing promising alternatives for the treatment of diseases caused by this protozoan.

Toxicological assessment of the Achyrocline satureioides aqueous extract in the Caenorhabditis elegans alternative model.

Achyrocline satureioides, popularly called "marcela" in Brazil, is used in traditional medicine in South America. A. satureioides, inflorescences are used for many conditions, including to minimize the Sars-Cov-2 symptoms. Therefore, the aim of this study was to determine the toxicity profile of A. satureioides aqueous extract (ASAE), using the Caenorhabditis elegans (C. elegans) alternative model. Survival, reproduction, development, and transgenerational assays were performed. The effects of ASAE were investigated under conditions of thermal stress and presence of oxidant hydrogen peroxide (H2O2). In addition, C. elegans strains containing high antioxidant enzyme levels and elevated lineages of daf-16, skn-1 and daf-2 regulatory pathways were examined. The ASAE LC50 value was found to be 77.3 ± 4 mg/ml. The concentration of ASAE 10 mg/ml (frequently used in humans) did not exhibit a significant reduction in worm survival at either the L1 or L4 stage, after 24 or 72 hr treatment. ASAE did not markedly alter the body area. In N2 strain, ASAE (10 or 25 mg/ml) reversed the damage initiated by H2O2. In addition, ASAE protected the damage produced by H2O2 in strains containing significant levels of sod-3, gst-4 and ctl - 1,2,3, suggesting modulation in these antioxidant systems by this plant extract. ASAE exposure activated daf-16 and skn-1 stress response transcriptional pathways independently of daf-2, even under extreme stress. Data suggest that ASAE, at the concentrations tested in C. elegans, exhibits a reliable toxicity profile, which may contribute to consideration for safe use in humans.

Global prevalence of potentially pathogenic free-living amoebae in sewage and sewage-related environments-systematic review with meta-analysis.

Free-living amoebae (FLA) include amphizoic microorganisms important in public health, widely isolated from air, water, and soil. However, its occurrence in sewage-related environments still needs to be systematically documented. This study summarizes the occurrence of FLA in sewage-related environments through a systematic review with meta-analysis. A total of 1983 scientific article were retrieved from different databases, of which 35 were selected and analyzed using a random effects forest plot model with a 95% confidence interval (IC). The pooled overall prevalence of FLA in sewage across 12 countries was 68.96% (95% IC = 58.5-79.42). Subgroup analysis indicates high prevalence in all environments analyzed, including sewage water from the sewage treatment plant (81.19%), treated sewage water (75.57%), sewage-contaminated water (67.70%), sediment contaminated by sewage (48.91%), and sewage water (47.84%). Prevalence values of Acanthamoeba spp., Hartmanella/Vermamoeba spp., and Naegleria spp. are 47.48%, 28.24%, and 16.69%, respectively. Analyzing the species level, the distribution is as follows: Acanthamoeba palestinensis (88%), A. castellanii (23.74%), A. astronyxis (19.18%), A. polyphaga (13.59%), A. culbertsoni (12.5%), A. stevensoni (8.33%), A. tubiashi (4.35%) and A. hatchetti (1.1%), Naegleria fowleri (28.4%), N. gruberi (25%), N. clarki (8.33%), N. australiensis (4.89%) and N. italica (4.29%), Hartmannella/Vermamoeba exundans (40%) and H.V. vermiform (32.61%). Overall, our findings indicate a high risk associated with sewage-related environments, as the prevalence of FLA, including pathogenic strains, is high, even in treated sewage water. The findings of this study may be valuable both for risk remediation actions against amoebic infections and for future research endeavors.

Agar dehydration: a simple method for long-term storage of Acanthamoeba spp. collection at room temperature.

This study describes dehydration of agar containing cysts as a novel and inexpensive method for long-term storage of Acanthamoeba spp. collections at room temperature. Five hundred microliters of axenically cultured Acanthamoeba spp. trophozoites (106 cells/mL) in PYG media or 150 µl of amoeba suspension (106 cells or cysts/mL) from monoxenic plate culture was spread onto the surface of non-nutritive agar (NNA, 2-3-mm thick) without or with a layer of heat-inactivated Escherichia coli, respectively. The plates were sealed and incubated at 30 °C. After the encystment, the Parafilm® was removed, and the plates were kept at the same temperature until the NNA was completely dehydrated. The dehydrated cyst-containing NNA was cut in rectangles and stored in airtight tubes at room temperature for up to 3 years. Cyst viability was assessed by inoculating them in fresh NNA with a layer of E. coli and in PYG followed by incubation at 30 °C. One hundred percent of samples from all specimens (19) stored over the 3 years allowed new cultures to be re-established; however, two strains showed reduced viability, at 66.7% and 62.5%, after 2 years of room temperature storage. One hundred percent of the cyst samples produced axenically and maintained in dry NNA allowed the re-establishment of axenic cultures through direct incubation in PYG, with excystment occurring within 24 or 48 h. For the first time, we report the dehydration of cyst-containing agar as an economical and effective method for the long-term storage of Acanthamoeba spp. collections at room temperature. It enables the creation of large collections using reduced space and economical transport of Acanthamoeba strains, in addition to allowing better organization of the collection.

A challenge in washing water with the sun: 24h of SODIS fails to inactivate Acanthamoeba castellanii cysts and internalized Pseudomonas aeruginosa under strong real sun conditions.

Despite access to drinking water being a basic human right, the availability of safe drinking water remains a privilege that many do not have and as a result, many lives are lost each year due to waterborne diseases associated with the consumption of biologically unsafe water. To face this situation, different low-cost household drinking water treatment technologies (HDWT) have been developed, and among them is solar disinfection (SODIS). Despite the effectiveness of SODIS and the epidemiological gains being consistently documented in the literature, there is a lack of evidence of the effectiveness of the batch-SODIS process against protozoan cysts as well as their internalized bacteria under real sun conditions. This work evaluated the effectiveness of the batch-SODIS process on the viability of Acanthamoeba castellanii cysts, and internalized Pseudomonas aeruginosa. Dechlorinated tap water contaminated with 5.6 × 103 cysts/L, contained in PET (polyethylene terephthalate) bottles, was exposed for 8 h a day to strong sunlight (531-1083 W/m2 of maximum insolation) for 3 consecutive days. The maximum water temperature inside the reactors ranged from 37 to 50 °C. Cyst viability was assessed by inducing excystment on non-nutrient agar, or in water with heat-inactivated Escherichia coli. After sun exposure for 0, 8, 16 and 24 h, the cysts remained viable and without any perceptible impairment in their ability to excyst. 3 and 5.5 log CFU/mL of P. aeruginosa were detected in water containing untreated and treated cysts, respectively, after 3 days of incubation at 30 °C. The batch-SODIS process is unable to inactivate A. castellanii cysts as well as its internalized bacteria. Although the use of batch SODIS by communities should continue to be encouraged, SODIS-disinfected water should be consumed within 3 days.

First report of free-living amoebae in sewage treatment plants in Porto Alegre, southern Brazil.

Free-living amoebae (FLA) are amphizoic protozoans with a cosmopolitan distribution. Some strains of species are associated with infections in humans. They feed on microorganisms by phagocytosis; however, some of these can become endocytobionts by resisting this process and taking shelter inside the amoeba. The whole world is experiencing increasing shortage of water, and sewage is being reused, so the study of this environment is important in public health context. The objective of this work was to identify FLA present in sewage treatment plants in Porto Alegre, Brazil. About 1 L samples were collected from eight stations (raw and treated sewage) in January, February, July, and August 2022. The samples were sown in monoxenic culture, and the isolated amoebae were subjected to morphological and molecular identification. Polymerase chain reaction results indicated the presence of the genus Acanthamoeba in 100% of the samples. Gene sequencing showed the presence of Acanthamoeba lenticulata and Acanthamoeba polyphaga - T5 and T4 genotypes - respectively, which are related to pathogenicity. The environment where the sewage is released can be used in recreational activities, exposing individuals to potential interactions with these amoebae and their potential endocytobionts, which may pose risks to public health.

First report of free-living amoebae in watercourses in southern Brazil: molecular diagnosis and phylogenetic analysis of Vermamoeba vermiformis, Naegleria gruberi, and Acanthamoeba spp.

Free-living amoebae (FLA) are protozoa dispersed in different environments and are responsible for different infections caused to humans and other animals. Microorganisms such as Acanthamoeba spp., Vermamoeba sp., and Naegleria sp. are associated with diseases that affect the central nervous system, in addition to skin infections and keratitis, as occurs in the genus Acanthamoeba and with Vermamoeba vermiformis. Due to the concerns of these FLA in anthropogenic aquatic environments, this work aimed to identify these microorganisms present in waters of Porto Alegre, Brazil. One litre sample was collected in two watercourses during the summer of 2022 and inoculated onto non-nutrient agar plates containing heat-inactivated Escherichia coli. Polymerase chain reaction results indicated the presence of FLA of the genera Acanthamoeba, Vermamoeba, and Naegleria in the study areas. Genetic sequencing indicated the presence of V. vermiformis and Naegleria gruberi. These aquatic and anthropogenic environments can serve as a means of spread and contamination by FLA, which gives valuable information on public health in the city.

Occurrence, molecular diversity and pathogenicity of Acanthamoeba spp. isolated from aquatic environments of Northeastern Brazil.

Acanthamoeba is a free-living amoeba (FLA) that is ubiquitous in nature and can cause serious pathologies in humans. This protozoan has been detected in several environmental sources, such as soil, water, and swimming pools. The aim of this study was to evaluate the occurrence and molecular diversity of Acanthamoeba spp. in aquatic environments of the state of Sergipe, northeastern Brazil, and to determine the pathogenic potential of the isolated samples. A total of 138 samples were collected from 69 aquatic environments and, after cell culture, 74% of the samples were positive for FLA, 47% belonging to the genus Acanthamoeba. Genotypic analysis was performed using the primers JDP1 and JDP2, confirming distinct Acanthamoeba genotypes: 18 (75%) isolates belonging to genotype T4, two (8%) to T3, and one isolate (4%) to genotype T5. Tests carried out to analyze the pathogenic potential showed that 11 isolates could grow at 0.5 M mannitol concentration and seven isolates supported hyperosmolarity. In the thermotolerance test, two isolates grew at 37°C. These results confirm the presence and the pathogenic potential of FLA of the genus Acanthamoeba in aquatic environments of the municipalities of Sergipe.

Epidemiological and immunological gains from solar water disinfection: Fact or wishful thinking?

OBJECTIVE: There is still no consensus on the impact of using solar disinfection (SODIS) to reduce the prevalence of waterborne gastrointestinal diseases. The reported reduction in diarrhoea prevalence among SODIS users has been attributed to the consumption of water free of viable pathogens. However, it has also been suggested that ingestion of SODIS-inactivated pathogens may induce protective immunological changes that may also contribute to a reduction in the frequency of diarrhoea. The present study aimed to critically review the epidemiological and immunological gains of using SODIS. METHODS: We critically reviewed 22 articles published in English, selected from 2118 records systematically retrieved from the databases. RESULTS: All trials (except one) reported a significant reduction in diarrhoea prevalence among children using SODIS, but some of the data from trials report contrary findings. All in vitro and in vivo assays indicate that SODIS-inactivated pathogenic bacteria have the potential to induce immunological alterations that may result in protective immunological effects. Studies with a low risk of bias are still awaited to confirm the ability to use SODIS to reduce the prevalence of diarrhoea. CONCLUSION: Reducing the prevalence of diarrhoea depends on the success of SODIS delivery strategies in inducing behavioural changes in communities that result in the production of SODIS-compliant outcomes. The results of trials reporting a reduction in the prevalence of diarrhoea due to the use of SODIS seem to support the hypothesis of the contribution of the protective immunological effect against diarrhoea in SODIS users.

Prevalence of free-living amoebae in swimming pools and recreational waters, a systematic review and meta-analysis.

Free-living amoebae (FLA) are cosmopolitan microorganisms known to be pathogenic to humans who often have a history of contact with contaminated water. Swimming pools and recreational waters are among the environments where the greatest human exposure to FLA occurs. This study aimed to determine the prevalence of FLA in swimming pools and recreational waters, through a systematic review and meta-analysis that included studies published between 1977 and 2022. A total of 106 studies were included and an overall prevalence of FLA in swimming pools and recreational waters of 44.34% (95% CI = 38.57-50.18) was found. Considering the studies published up to 2010 (1977-2010), between 2010 and 2015, and those published after 2010 (> 2010-2022), the prevalence was 53.09% (95% CI = 43.33-62.73) and 37.07% (95% CI = 28.87-45.66) and 45.40% (95% CI = 35.48-55.51), respectively. The highest prevalence was found in the American continent (63.99%), in Mexico (98.35%), and in indoor hot swimming pools (52.27%). The prevalence varied with the variation of FLA detection methods, morphology (57.21%), PCR (25.78%), and simultaneously morphology and PCR (43.16%). The global prevalence by genera was Vahlkampfia spp. (54.20%), Acanthamoeba spp. (33.47%), Naegleria spp. (30.95%), Hartmannella spp./Vermamoeba spp. (20.73%), Stenamoeba spp. (12.05%), and Vannella spp. (10.75%). There is considerable risk of FLA infection in swimming pools and recreational waters. Recreational water safety needs to be routinely monitored and, in case of risk, locations need to be identified with warning signs and users need to be educated. Swimming pools and artificial recreational water should be properly disinfected. Photolysis of NaOCl or NaCl in water by UV-C radiation is a promising alternative to disinfect swimming pools and artificial recreational waters.

Clinical and molecular diagnosis of Acanthamoeba keratitis in contact lens wearers in southern Brazil reveals the presence of an endosymbiont.

Acanthamoeba keratitis (AK) is an infection that is mostly observed in contact lens wearers. It is often misdiagnosed causing delays in the administration of the correct treatment. The aim of this study was to report the outcome of clinical and molecular diagnosis of AK cases during the summer of 2019 in the southern region of Brazil. Three suspected cases of AK were discovered after an ophthalmic examination at a public hospital in the city of Porto Alegre. These cases were then confirmed through laboratory diagnosis (cell culture and molecular analysis by PCR and sequencing). In each of the three clinical sample cell cultures of corneal scraping and molecular analysis confirmed the presence of Acanthamoeba spp., all belonging to the morphological group II and to the genotype T4, which is the most common genotype associated with AK. In addition, Acanthamoeba spp. isolated from one of the clinical samples was found to harbor the Candidatus Paracaedibacter acanthamoeba, a bacterial endosymbiont. The presence of Ca. Paracaedibacter acanthamoeba in clinical isolates requires further research to reveal its possible role in the pathogenicity of Acanthamoeba infections.

Genotype distribution of Acanthamoeba in keratitis: a systematic review.

Acanthamoeba spp. are among the most worldwide prevalent protozoa. It is the causative agent of a disease known as Acanthamoeba keratitis, a painful and severe sight-threatening corneal infection that can lead to blindness. In recent years, the prevalence of Acanthamoeba keratitis has rapidly increased, growing its importance to human health. This systematic review aims to assess the distribution of Acanthamoeba sp. genotypes causing keratitis around the world, considering the sample collected type and the used identification method. Most of the cases were found in Asia and Europe. Not surprisingly, the T4 genotype was the most prevalent worldwide, followed by T3, T15, T11, and T5. Furthermore, the T4 genotype contains a higher number of species. Given the differences in pathology, susceptibility to treatment, and clinical outcome between distinct genotypes, it is essential to genotype isolates from Acanthamoeba keratitis cases to help to establish a better correlation between in vitro and in vivo activities, resulting in better drug therapies and successful treatment in cases of this important ocular infection.

Single cell cloning of free-living amoebae with isolated square agar.

An alternative amoeba cloning technique is described. The amoebic cells were scraped from the agar and inoculated in a new agar medium separated in isolated squares, preventing a cell from invading the space of another cell, increasing the reliability of cloning, and providing an efficient quality control of technique.

Naegleria fowleri-associated meningoencephalitis in a cow in Southern Brazil-first molecular detection of N. fowleri in Brazil.

Naegleria fowleri, a free-living and thermophilic ameba, is the etiological agent of primary amebic meningoencephalitis (PAM). PAM is a rare and highly fatal neurologic disease in humans, and has been rarely documented in animal species. This report describes the pathological and etiological findings of a fatal case of N. fowleri-associated meningoencephalitis in a cow in Southern Brazil. Microscopic findings were consistent with severe, multifocal, hemorrhagic, and necrosuppurative meningoencephalitis associated with a large number of amebic trophozoites compatible with N. fowleri. Brain samples subjected to molecular assays generated a 315 bp fragment, which presented 99% identity with a N. fowleri sequence previously deposited in GenBank. This is the first study reporting the molecular detection of N. fowleri in a case of cattle meningoencephalitis in Latin America, and the obtained sequence represents the first GenBank deposit of N. fowleri identified in Brazil to this day. Additionally, the case reported is the second occurrence of N. fowleri-associated disease in the same city, drawing attention to the local importance of infection by this ameba and potential risk for human infections.

Free-living amoebae and their relationship to air quality in hospital environments: characterization of Acanthamoeba spp. obtained from air-conditioning systems.

Free-living amoebae (FLA) are widely dispersed in the environment, can cause opportunistic and non-opportunistic infections in humans and other animals. The aim of the present study was characterize FLA obtained from air-conditioners of a public hospital in the city of Florianópolis, SC, Brazil. Fifty-four dust samples were collected of air conditioners, and were inoculated on 1.5% non-nutrient agar, overlaid with layers of Escherichia coli. Subsequently the isolates were axenised in PYG growth medium. The morphological and molecular characterization of the isolates was performed, as well as the tolerance (physiological) assays were used to evaluate the pathogenic potential. The results revealed the presence of FLA in 42 (77.8%) of the collected samples. Of these, 39 (92.9%) axenic isolates of FLA were obtained for morphological and genotypic studies. All the isolates characterized belong to the genus Acanthamoeba. Nineteen (48.7%) isolates belong to the genotype T4, 16 (41.0%) to the T5 genotype and 4 (10.3%) to genotype T11. Seven (18.0%) isolates were considered potentially pathogenic in tolerance assays. These findings require attention, considering the isolation environment and immunocompromised characteristics of many hospitalized patients.

Acanthamoeba keratitis in Porto Alegre (southern Brazil): 28 cases and risk factors.

The increasing use of contact lenses worldwide has led to an increase in cases of Acanthamoeba keratitis, which are often associated with inappropriate cleaning of contact lenses and lens cases. This study aimed to retrospectively review 28 cases of Acanthamoeba keratitis in Porto Alegre (southern Brazil) and identify the risk factors and clinical outcomes of affected patients. Most patients had higher education (66.6%), all were users of contact lenses, mostly women (67.9%). Most patients were soft contact lens wearers (66.7%) and 85.7% used multipurpose cleaning solutions. Sixteen patients (64.0%) used to wear contact lenses while swimming and/or bathing. Pain was the most common symptom (92.6%). For treatment, patients used polyhexamethylene biguanide drops (92.6%), propamidine isethionate drops (81.5%), chlorhexidine drops (55.6%), topical corticosteroids (63.0%), and systemic corticosteroids (37.0%). Herpes simplex keratitis was the most common misdiagnosis (72.7%). The majority of patients (76.0%) underwent a corneal transplant to control the disease.

Contact lens-related polymicrobial keratitis: Acanthamoeba spp. genotype T4 and Candida albicans.

A 31-year-old female daily user of contact lenses sought medical attention, reporting blurred vision and irritation of the left eye. Slit-lamp examination revealed hyperemia and an irregular corneal epithelium surface, and empirical treatment was started. A corneal scrape was obtained and examined for the presence of fungi, bacteria, and Acanthamoeba spp. The results of the microbial culture revealed growth of Acanthamoeba spp. and Candida albicans. The Acanthamoeba isolate was characterized by cyst morphology as belonging to group II according to Pussard and Pons. Sequencing of the diagnostic fragment 3 (DF3) region located on the 18S ribosomal DNA identified the isolate as genotype T4. The patient was treated with chlorhexidine 0.02% and polyhexamethylene biguanide (PHMB) 0.02% drops for 5 months until the infection resolved. Lately, rare cases of polymicrobial keratitis associated with Acanthamoeba and Candida albicans have been reported. Cases of co-infection are more difficult to treat, since the specific treatment depends on precise identification of the agents involved.

Interaction Between Methicillin-Resistant Staphylococcus aureus (MRSA) and Acanthamoeba polyphaga.

The interactions that occur between bacteria and amoebae can give through mutual relations, where both organisms benefit from the association or parasitic in which one organism benefits at the expense of the other. When these organisms share the same environment, it can result in some changes in the growth of organisms, in adaptation patterns, in morphology, development or even in their ability to synthesize proteins and other substances. In this study, the interaction between Acanthamoeba polyphaga and Staphylococcus aureus (MRSA) was evaluated using a co-culture model at different incubation times. The results showed that 89% of amoebic cells remained viable after contact with the bacteria. The bacterial isolate was visualized inside the amoeba through confocal microscopy and fluorescence for up to 216 h of co-cultivation. The lysate of amoebic culture increased the growth of S. aureus (MRSA), and the effect of supernatant of culture inhibited bacterial growth over the incubation times, suggesting that A. polyphaga produced some metabolite, that inhibited the growth of bacteria. Moreover, the encystment of the A. polyphaga was increased by the bacteria presence. The results show that A. polyphaga and S. aureus interaction may have an important influence on survival of both, and specially indicate a possible effect on the metabolics characteristics each other.

Nanoemulsions Containing a Coumarin-Rich Extract from Pterocaulon balansae (Asteraceae) for the Treatment of Ocular Acanthamoeba Keratitis.

This study describes the incorporation of a coumarin-rich extract from Pterocaulon balansae into nanoemulsions intended for the local treatment of ocular keratitis caused by Acanthamoeba. The n-hexane dewaxed extract of P. balansae was characterized by HPLC/PDA and UPLC/MS. The presence of four major coumarins was detected, where 5-methoxy-6,7-methylenedioxycoumarin was selected as a chemical marker. This extract was then incorporated into nanoemulsions composed of medium chain triglycerides and egg-lecithin, through spontaneous emulsification. Such a procedure yielded the formation of monodisperse nanoemulsions in a sub-300-nm range, regardless of the amount of extract incorporated (1.0-5.0 mg/mL). The amoebicidal activity against Acanthamoeba castellanii was both dose-dependent and incubation time-dependent. A reduction of 95% of trophozoite viability was detected after 24 h of incubation with a nanoemulsion at 1.25 mg/mL of coumarins, being a similar effect detected for chlorhexidine. These results suggest a potential of the formulations developed in this study as a new strategy for the treatment of ocular keratitis caused by Acanthamoeba.

Evaluation of the immunodiagnostic potential of a recombinant surface protein domain from Acanthamoeba castellanii.

Acanthamoeba spp. are free-living protists widely distributed in environment, able to cause keratitis, encephalitis and skin lesions in humans and animals. Acanthamoeba spp. exist in two forms: an infective trophozoite and a dormant cyst. Several factors contribute to the pathogenesis of Acanthamoeba spp. The parasite adhesion to the host cell is the primary step for infection and is mediated by a mannose binding-protein, expressed in the surface and considered the main pathogenicity factor in Acanthamoeba spp. So far, there was no evidence of another surface protein of Acanthamoeba spp. relevant for host invasion or infection by these organisms. The aims of this study were to identify and characterize an Acanthamoeba castellanii surface protein and to evaluate its diagnostic potential. In silico predictions of surface proteins allowed to identify the A. castellanii calreticulin as a possible surface antigen. The coding sequence of a predicted extracellular domain of A. castellanii calreticulin was cloned by in vivo homologous recombination and the recombinant polypeptide (AcCRT29-130) was produced. Its immunodiagnostic potential was assessed in a recombinant antigen-based ELISA with sera from experimentally infected rats that developed keratitis and encephalitis, and sera from patients with encephalitis. The AcCRT29-130 was significantly more recognized by sera from encephalitis infected rats in comparison with the non-infected controls. Human sera from encephalitis patients, however presented no significant response. These results showed the AcCRT29-130 potential for A. castellanii infection immunodiagnosis in animals, with further studies being required for assessment of its use for human infections.