Molecular characterization and epidemiology of Toxoplasma gondii isolates from free-range chickens in the southwest region of Goiás: new genotypes.

The purpose of this study was to isolate Toxoplasma gondii from tissues of free-range chickens in the southwestern region of Goiás, to detect and molecularly characterize the genetic material of the parasite, and to determine the seroprevalence of the protozoan parasite in these animals. A seroprevalence of T. gondii antibodies of 76% (19/25) was found among the chickens, while genetic material from their tissues was detected in 56% (14/25). A total of 14 isolates was obtained in the bioassay, ten of which were considered acute, eight were considered isolates of high virulence lethal to mice, and four of low virulence, considered non-lethal but with the ability to chronify the infection. Seven of the ten isolates showed significant morphometric differences from the RH strain, in terms of nucleus-complex-apical distance, length and width. Genotyping of the acute isolates was performed by RFLP-PCR, using 11 genetic markers: SAG1, SAG2 (3'SAG2 and 5'SAG2), alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and APICO. The results were compared and classified according to the genotypes listed on the ToxoDB Platform, where different profiles were observed indicating the presence of two known genotypes (#7 and #63) and five new genotypes (NEW 3, NEW4, NEW5, NEW6, NEW 7). The results showed high seroprevalence, isolation rate, molecular detection and genotypic variations of T. gondii in free-range chickens in the southwestern region of Goiás.

Molecular characterization of Cryptosporidium in ruminants and observation of natural infection by Cryptosporidium andersoni in sheep from Paraná, Brazil.

The aim of this study was to identify Cryptosporidium species found in cattle and sheep in Paraná, southern region of Brazil. Individual fecal samples from 458 bovines and 101 sheep were submitted for molecular analysis by PCR and nested PCR using specific primers for sequences of the 18S ribosomal unit (rRNA). Positive samples were analyzed using restriction fragment length polymorphism (RFLP), followed by genetic sequencing for species confirmation. The occurrence of Cryptosporidium was 11.27% (63/559). The highest occurrence was detected in lambs (12/59, 20.33%). From the 63 positive samples, it was possible to identify the species in 58 of them by RFLP and genetic sequencing. Five species of Cryptosporidium were identified: Cryptosporidium andersoni, Cryptosporidium bovis, Cryptosporidium ryanae, Cryptosporidium xiaoi, and Cryptosporidium parvum. The most prevalent species was C. andersoni (41.38%) and the least predominant was C. parvum (10.34%). The most abundant species of Cryptosporidium in dairy calves were C. andersoni (11/25) and C. ryanae (6/25). Of the 17 positive sheep, nine (52.94%) were infected with C. andersoni. This finding is the first report on the occurrence of C. andersoni in naturally infected sheep in Brazil and the first observation of a high absolute occurrence of this Cryptosporidium species in sheep.

SEROLOGICAL SURVEY FOR SELECTED PATHOGENS IN FREE-RANGING COUGARS (PUMA CONCOLOR) IN SÃO PAULO, BRAZIL.

This study performed a serological assay to assess the exposure of free-ranging cougars (Puma concolor) to four selected infectious agents, including Toxoplasma gondii, Leptospira spp., the feline immunodeficiency virus (FIV), and the feline leukemia virus (FeLV). Serum samples were collected from 27 free-ranging cougars along the Tietê River Basin, in the central region of the State of São Paulo, Brazil. The presence of antibodies against T. gondii was detected in 59.3% (16/27) of the serum samples through the modified agglutination test (MAT-t), which was the most prevalent agent. The microscopic agglutination technique (MAT-1) was used to investigate the occurrence of anti-Leptospira spp. antibodies, showing that 11.1% (3/27) of the sampled cougars were seropositive. The only serovar detected was Djasiman (L. interrogans). A commercial enzyme-linked immunosorbent assay (ELISA) licensed for use in domestic felines was used to investigate the occurrence of retroviruses. The ELISA test kits detected a prevalence of 11.1% (3/27) of FIV antibodies, while none of the samples tested showed any evidence of FeLV antigen. These results suggest that free-ranging cougars are exposed to potentially pathogenic agents. This study presented the first recorded occurrence of the serovar Djasiman in P. concolor.

Seroprevalence and systemic immune biomarkers associated with Toxoplasma gondii infection in blood donors from Southern Brazil.

The high seroprevalence of Toxoplasma gondii infection in Blood Banks could be a potential risk for contamination of blood recipients. The discovery of new biomarkers may help to distinguish between seropositive and seronegative donors. This study determined the seroprevalence and profile of systemic immune biomarkers associated with Toxoplasma gondii infection among blood donors from Southern Brazil. Peripheral blood was collected from 510 blood donors (52.2 % male; mean age: 36.61), 310, and 200 from Erechim, and Chapecó municipalities, respectively. Specific Toxoplasma gondii IgG and IgM antibodies were detected by Eletrochemioluminescence. Nested PCR and qPCR were performed to detectToxoplasma gondii DNA. Twenty-seven inflammatory factors were analyzed using a high-performance Luminex assay. Among 310 blood donors from Erechim, 44.5 % (138/310) were IgM(-)/IgG(+), and 1.3 % (4/310) were IgM(+)/IgG(+), while out of 200 blood donors from Chapeco, 42.5 % (85/200) were IgM(-)/IgG(+), and 2 % (4/200) were IgM(+)/ IgG(+). We did not find Toxoplasma gondii DNA in the samples analyzed by Nested PCR and qPCR.Additionally, IgM(-)/IgG(+) donors presented higher levels ofdistinct systemic mediators, and were indicated to be high producers of several systemic mediators (CCL11, CCL2, CCL3, CCL4, CXCL10, IL-1ß, IL-17, IFN-γ, IL-4, IL-9, IL-13, IL-10, IL-1Ra, vascular endothelial growth factor/VEGF, platelet-derived growth factor/PDGF, granulocyte-macrophage colony-stimulating factor/GM-CSF, and IL-7). However, IgM(+)/IgG(+) donors were found as high producers of CXCL8, CXCL10, CCL4, IL-1ß, IL-1Ra, IL-9, IL-13, and PDGF, while IgM(-)/IgG(-) donors showed unaltered levels for the most soluble mediators evaluated. These distinct biomarker signatures might help identify potential factors to distinguish between IgM(-) and IgM(+) donors.

High occurrence of viable forms of Cryptosporidium and Giardia in domestic sewage from an agricultural region of Brazil.

Cryptosporidium and Giardia are the main etiologies of waterborne outbreaks caused by protozoa. These parasites are commonly detected in wastewater; however, there is little knowledge about the concentration of viable forms in treated sewage, mainly in small communities. To understand more about the presence of viable oocysts and cysts in domestic sewage, we monitored the affluent and effluent of a wastewater treatment plant (WWTP) in inner-city Brazil. Ten samplings and seven follow-ups were performed in 2020. Samples were concentrated by centrifugation, filtration and purified by fluctuation. Viability was accessed by propidium-monoazide (PMA) associated with nPCR and qPCR. Both viable protozoa were detected in all raw sewage samples (average: 438.5 viable oocysts/L). Regarding treated sewage, Cryptosporidium was detected in all of the samples (average: 92.8 viable oocysts/L) and Giardia was detected in 70% with viable cysts in 30%. Considering the follow-ups, 31.17% of Cryptosporidium viable oocysts remained in the effluent after the treatment. High amounts of Cryptosporidium and a high frequency of Giardia were detected, therefore both arrived at WWTP and were discharged into the river. These alert the presence of agro-industrial effluents into domestic sewage and demonstrated the effectiveness of the concentration technique for monitoring protozoa in wastewater.

Moderate intestinal immunopathology after acute oral infection with Toxoplasma gondii oocysts is associated with expressive levels of serotonin.

BACKGROUND: Invasion of the intestinal mucosa by T. gondii elicits a local immune response of variable intensity. These reactions can be lethal in C57BL/6 mice. The tissue damage caused by inflammation and the functional effects depend on the host immunity, strain, and developmental form of the parasite. We investigated the effects of acute oral infection with T. gondii on histoarchitecture, enteric nervous system (ENS), and inflammatory markers in the jejunum and ileum of mice. METHODS: Female C57BL/6 mice were divided into a control group and a group orally infected with 1000 sporulated T. gondii oocysts (ME-49 strain). After 5 days, jejunum and ileum were collected and processed for analyzes (e.g., histological and histopathological examinations, ENS, cytokine dosage, myeloperoxidase, nitric oxide activity). MAIN RESULTS: In infected mice, we observed a significant increase in serotonin-immunoreactive cells (5-HT IR) in the intestinal mucosa, as well as cellular infiltrates in the lamina propria, periganglionitis, and ganglionitis in the myenteric plexus. We also noted decreased neuron density in the jejunum, increased population of enteric glial cells in the ileum, histomorphometric changes in the intestinal wall, villi, and epithelial cells, remodeling of collagen fibers, and increased myeloperoxidase activity, cytokines, and nitric oxide in the intestine. CONCLUSIONS AND INFERENCES: Acute infection of female mice with T. gondii oocysts resulted in changes in ENS and a marked increase in 5-HT. These changes are consistent with its modulatory role in the development of moderate acute inflammation. The use of this experimental model may lend itself to studies aimed at understanding the pathophysiological mechanisms of intestinal inflammation in humans involving ENS.

First report of Giardia duodenalis assemblage F in humans and dogs in southern Brazil.

The present study aimed to molecularly characterize Giardia duodenalis from stool samples of humans, dogs, and cats. Molecular analyses were performed on 59 samples that tested positive for G. duodenalis on coproparasitological examinations. After extraction, the samples were first tested by nested polymerase chain reaction (n-PCR) analysis of the SSU-rRNA gene, and for the samples that were positive, the ß-giardin, TPI, and GDH genes were analyzed. The amplicons obtained in the n-PCR of the ß-giardin gene were subjected to PCR-restriction length polymorphism (RFLP) analysis and subsequent digestion with the enzyme HaeIII to differentiate the assemblages. Seven (11.8 %), 34 (57.7 %), and 18 (30.5 %) out of 59 samples were from humans, dogs, and cats, respectively. Nested-PCR results showed that 49.2 % (29/59) of samples were positive for the SSU-rRNA gene, with 42.9 % (3/7) of humans, 55.9 % (19/34) of dogs, and 38.9 % (7/18) of catsve. Of the other genes analyzed, ß-giardin was amplified most frequently, in 34.5 % (10/29) of samples, followed by GDH in 27.6 % (8/29) of samples, and TPI in 10.3 % (3/29) of samples. Only one sample from a dog showed the amplification of all genes. PCR-RFLP analysis showed assemblage F in a human, dog, and cat samples; and assemblage C and D in dog samples. This is the first description of assemblage F in humans from Brazil and the first description of assemblage F in dogs. Further studies are needed to verify the frequency with which these infections occur, and provide information that will contribute to the molecular epidemiological understanding of giardiasis.

Biogenic silver nanoparticles (AgNp-Bio) restore testosterone levels and increase TNF-α and IL-6 in Leydig cells infected with Toxoplasma gondii.

Toxoplasma gondii, a protozoan parasite, is responsible for toxoplasmosis. The available therapy for patients with toxoplasmosis involves a combination of pyrimethamine and sulfadiazine, which have several adverse effects, including bone marrow suppression, megaloblastic anemia, leukopenia, and granulocytopenia. The development of therapeutic alternatives is essential for the management of toxoplasmosis, emphasizing the recent advances in nanomedicine. This study aimed to evaluate the in vitro effects of biogenic silver nanoparticles (AgNp-Bio) on tachyzoite forms and Leydig cells infected with T. gondii. We observed that the AgNp-Bio reduced the viability of the tachyzoites and did not exhibit cytotoxicity against Leydig cells at low concentrations. Additionally, treatment with AgNp-Bio reduced the rate of infection and proliferation of the parasite, and lowered the testosterone levels in the infected cells. It increased the levels of IL-6 and TNF-α and reduced the levels of IL- 10. Among the morphological and ultrastructural changes, AgNp-Bio induced a reduction in the number of intracellular tachyzoites and caused changes in the tachyzoites with accumulation of autophagic vacuoles and a decrease in the number of tachyzoites inside the parasitophorous vacuoles. Collectively, our data demonstrate that the AgNp-Bio affect T. gondii tachyzoites by activating microbicidal and inflammatory mechanisms and could be a potential alternative treatment for toxoplasmosis.

Biogenic silver nanoparticles reduce Toxoplasma gondii infection and proliferation in RAW 264.7 macrophages by inducing tumor necrosis factor-alpha and reactive oxygen species production in the cells.

Owing to the serious adverse effects caused by pyrimethamine and sulfadiazine, the drugs commonly used to treat toxoplasmosis, there is a need for treatment alternatives for this disease. Nanotechnology has enabled significant advances toward this goal. This study was conducted to evaluate the activity of biogenic silver nanoparticles (AgNp-Bio) in RAW 264.7 murine macrophages infected with the RH strain of Toxoplasma gondii. The macrophages were infected with T. gondii tachyzoites and then treated with various concentrations of AgNp-Bio. The cells were evaluated by microscopy, and culture supernatants were collected for ELISA determination of their cytokine concentration. Treatment with 6 µM AgNp-Bio reduced the infection and parasite load in infected RAW 264.7 macrophages without being toxic to the cells. The treatment also induced the synthesis of reactive oxygen species and tumor necrosis factor-alpha (both pro-inflammatory mediators), which resulted in ultrastructural changes in the tachyzoites and their intramacrophagic destruction. Our findings suggest that AgNp-Bio affect T. gondii tachyzoites by activating microbicidal and pro-inflammatory mechanisms and may be a potential alternative treatment for toxoplasmosis.

A systematic literature review and meta-analysis of Toxoplasma gondii seroprevalence in goats.

The aim of this study was evaluate to seroprevalence of Toxoplasma gondii in goats worldwide and the main risk factors associated from 2000 to 2020, through meta-analysis with 55,317 goats from 75 reports published in seven electronic databases of major global importance. A global seroprevalence detected was 27.49% (95% CI 24.15-30.95; I2 = 99%), with the lowest percentage in Asia (20.74%; 95% CI 16.45-25.39) and highest in Central America (62.15%; 95% CI 57.28-66.90) and Europe (31.53%; 95% CI 21.71-42.26). The seropositivity in Africa and South America were (29.41%; 95% CI 19.11-40.89) and (29.76%; 95% CI 25.84-33.83), respectively. The seroprevalence was associated with presence of cats (OR 2.22; 95% CI 1.30-3.82), goats older than one year (OR 1.77; 95% CI 1, 37-2.29), females (OR 1.43; 95% CI 1.23-1.65), rearing system (extensive vs. intensive) (OR 4.82; 95% CI 1.96-11, 84) and rearing system (semi-intensive vs. intensive) (OR 1.48; 95% CI 1.48-6.13). The heterogeneity was evidenced in most world regions and the risk factors may play roles in varying the seroprevalence.

Neospora caninum infection and reproductive problems in dairy cows from Brazil: A case-control study.

Neosporosis, an infectious disease caused by the protozoan Neospora caninum, has been associated with economic losses in cattle rearing worldwide. However, previous studies have not presented any evidence regarding the association between serological status of neosporosis and alteration of the reproductive parameters. Thus, this study aimed to determine whether N. caninum is associated with reproductive disorders and to evaluate the possible risk factors of the infection. Blood samples from 202 dairy cows, 51 with a history of reproductive disorders (case group) and 151 without (control group), were collected from different farms in Brazil. Epidemiological questionnaires were conducted with all the farmers. Serum samples were subjected to an indirect fluorescent antibody test to detect antibodies against the parasite. In total, 28.22% (57/202) of the cows were seropositive: 47.06% (24/51) from the case group and 21.85% (33/151) from the control group. By logistic regression, cows aged ≥48 months and cows with history of abortion were 4.8 (95% confidence interval [CI] = 1.91-12.05; p = 0.001) and 2.3 (95% CI = 1.06-5.1; p = 0.034) times more likely to be seropositive, respectively. Furthermore, our results show an association between N. caninum seropositivity and abortion in dairy cows from Brazil with poor management conditions and N. caninum seropositivity risk factors for reproductive disorders.

Prevalence and risk factors of Eimeria spp. natural infection in sheep from northern Paraná, Brazil.

The present study aimed to perform an epidemiological and morphological identification of Eimeria infection in sheep in Brazil. Fecal samples from sheep were collected from 20 farms in northern Paraná, Brazil. An epidemiological questionnaire was used to evaluate the risk factors. Fecal samples containing oocysts per gram of feces (OoPG) ≥1000 were subjected to the modified Willis-Mollay method to perform oocyst identification. Sporulated oocysts were observed microscopically for morphological identification. A total of 807 fecal samples were collected. Based on the morphological characteristics of the sporulated oocysts, 10 species of Eimeria were identified, with main species observed: Eimeira ovinoidalis (98.1%), Eimeria crandallis (87.6%), Eimeria parva (79.1%), and Eimeria bakuensis (60.8%). Only 2.6% (7/268) of the sheep were infected with a single species, 4.8% (13/268) contained two different species, and 92.5% (248/268) were infected with three or more species. The analysis of risk factors showed that an intensive rearing, no rotation of pasture, dirt, and slatted floors, and age up to 12 months were associated with infection. This study showed a high prevalence of Eimeria natural infection in sheep from northern Paraná, Brazil. Furthermore, based on the risk factors, good management and hygiene practices must be employed to avoid infection.

Seroprevalence of Neospora caninum and risk factors associated with infection in water buffaloes (Bubalus bubalis) from Maranhão State, Brazil.

The aim of this work was to determine the seroprevalence of Neospora caninum in buffaloes (Bubalus bubalis) from Maranhão State, Brazil, and identify risk factors associated with infection. In total, 338 buffaloes of different ages and both sexes from four farms were randomly selected. Information about the study region, animals and management was collected using an epidemiological questionnaire. Sera were subjected to an indirect fluorescent antibody test (IFAT) to detect anti-N caninum antibodies, while N. caninum DNA was detected using the polymerase chain reaction (PCR) assays. The overall seroprevalence of N. caninum in buffaloes was 27.5% (93/338), while DNA was detected in 3.0% (3/101) samples. The proportion of positive animals detected by IFAT and PCR simultaneously was 2.6% (2/77). The risk factors for N. caninum infection were contact with fomites (p = 0.022), management conditions (p = 0.005), calving interval of 20 months (p = 0.010) and deworming management (application 3 times a year in adults and calves, p = 0.020; change of anthelmintic group, p = 0.040). By multivariate analysis, management conditions was a risk factor for N. caninum infection with odds ratio of 2.2 (95% CI 1.0-4.6). This is the first report of the prevalence and risk factors for neosporosis in B. bubalis of Maranhão State, Brazil. Thus, N. caninum is widely distributed in buffalo herds in Maranhão, with management conditions being a risk factor for infection. Further studies should be conducted to elucidate the importance of buffaloes in the epidemiology of neosporosis in Maranhão State.

Ocular Disease in Mice Inoculated with Pork Heart Samples Infected with Toxoplasma gondii.

PURPOSE: We analyzed the frequency, viability, and genetic characteristics of T. gondii in pork heart samples. METHODS: Thirty-five fresh pork samples were purchased in a slaughterhouse in Erechim city. The DNA was extracted and qPCR was performed. T. gondii genotyping was performed using PCR-RFLP analysis. Positive samples were digested and inoculated in mice for viability analysis. RESULTS: Our results showed that T. gondii DNA was detected in 25.7% of the pork heart samples and genotyping revealed one new atypical strain. The viability analyses demonstrated that 40% of mice presented clinical signs of T. gondii infection. qPCR was positive in the lung, liver, and brain of mice that presented clinical signs of T. gondii infection. Also, the histopathology analysis showed retinal disorganization, retinal detachment, inflammatory cell infiltration, and fibrosis in the eyes analyzed. CONCLUSION: Our findings have shown that pork eat from southern Brazil may contain live T. gondii that could be associated with toxoplasmosis.

Prevalence and risk factors of Eimeria spp. natural infection in sheep from northern Paraná, Brazil / Prevalência e fatores de risco da infecção natural por Eimeira spp. em ovinos na região norte do Paraná, Brasil

Abstract The present study aimed to perform an epidemiological and morphological identification of Eimeria infection in sheep in Brazil. Fecal samples from sheep were collected from 20 farms in northern Paraná, Brazil. An epidemiological questionnaire was used to evaluate the risk factors. Fecal samples containing oocysts per gram of feces (OoPG) ≥1000 were subjected to the modified Willis-Mollay method to perform oocyst identification. Sporulated oocysts were observed microscopically for morphological identification. A total of 807 fecal samples were collected. Based on the morphological characteristics of the sporulated oocysts, 10 species of Eimeria were identified, with main species observed: Eimeira ovinoidalis (98.1%), Eimeria crandallis (87.6%), Eimeria parva (79.1%), and Eimeria bakuensis (60.8%). Only 2.6% (7/268) of the sheep were infected with a single species, 4.8% (13/268) contained two different species, and 92.5% (248/268) were infected with three or more species. The analysis of risk factors showed that an intensive rearing, no rotation of pasture, dirt, and slatted floors, and age up to 12 months were associated with infection. This study showed a high prevalence of Eimeria natural infection in sheep from northern Paraná, Brazil. Furthermore, based on the risk factors, good management and hygiene practices must be employed to avoid infection.

Resumo O presente estudo teve como objetivo realizar uma avaliação epidemiológica e morfométrica da infecção por Eimeria em ovinos no Brasil. Amostras fecais de ovinos foram coletadas em 20 fazendas no sul do Brasil. Um questionário epidemiológico foi utilizado para avaliar os fatores de risco. Amostras fecais, contendo oocistos por grama de fezes (OoPG) ≥1000, foram submetidas ao método de Willis-Mollay modificado para realizar a identificação de oocistos. Oocistos esporulados foram observados microscopicamente para identificação morfológica. Foram coletadas 807 amostras fecais. Com base nas características morfológicas e morfométricas dos oocistos esporulados, foram identificadas 10 espécies de Eimeria, com as principais espécies observadas: Eimeria ovinoidalis (98,1%), Eimeria crandallis (87,6%), Eimeria parva (79,1%) e Eimeria bakuensis (60,8%). Apenas 2,6% (7/268) dos ovinos estavam infectados com uma única espécie, 4,8% (13/268) continham duas espécies diferentes e 92,5% (248/268) estavam infectados com três ou mais espécies. A análise dos fatores de risco mostrou que uma criação intensiva, sem rotação de pasto, terra, piso de ripa e idade até 12 meses foram associadas à infecção. Este estudo mostrou uma alta prevalência de infecção natural por Eimeria em ovinos do norte do Paraná, Brasil. Além disso, com base nos fatores de risco, boas práticas de manejo e higiene devem ser empregadas para evitar infecções.

Seroprevalence and risk factors for Neospora caninum and Toxoplasma gondii in goats of Maranhão State, Brazil.

We estimated the seroprevalence and possible risk factors for neosporosis and toxoplasmosis in goats in the state of Maranhão, Brazil. In addition, the variables related to these animals and the management of the farm were investigated in terms of the significance of the associations. In total, 383 serum samples from goats, of both sexes and different ages, were collected from 15 farms in four municipalities. The indirect immunofluorescence test was used for antibody detection against Neospora caninum and Toxoplasma gondii. The overall seroprevalence of N. caninum in goats was 26.4% (101/382; IC 95% 22.3-31.1), and 114 out of 383 serum samples were T. gondii-seropositive (29.8%, IC 95% 25.4-34.5). In addition, the seroprevalence of coinfection of T. gondii and N. caninum in goats was 8.6% (33/382; IC 95% 6.2-11.8). The risk factors significantly associated with the seroprevalence of N. caninum were age, type of sheepfold floor, rearing system, feeding, pasture area cultivated, cats having access to the feed deposits, worming, slaughter place of the animals, history of abortion and the presence of dogs and cats. Regarding the seroprevalence of T. gondii infection, age, category, presence of other species and purpose of breeding were the risk factors. To our knowledge, this is the first report of the seroprevalence and risk factors for N. caninum and T. gondii in goats in the state of Maranhão, Brazil, which provides basic data for the implementation of strategies and control measures against neosporosis and toxoplasmosis.

Evaluation of quantitative polymerase chain reaction for the detection of Toxoplasma gondii oocysts shed by cats.

Felines are definitive hosts of Toxoplasma gondii and can shed oocysts in their feces, contaminating the environment. Sporulated oocysts are highly resistant to the environment and have higher infectivity, which are attributed to many toxoplasmosis outbreaks. The aim of the present study was to evaluate a quantitative polymerase chain reaction (qPCR) technique for the detection of T. gondii oocysts shed by cats. Twelve cats from a previous vaccine experiment were challenged orally with 600 cysts of the TgDoveBr8 strain on day 72. Fecal samples were collected daily using the centrifugal flotation technique, with microscopic examination (Sheather technique) and qPCR for 20 days after the challenge. Cats from all groups shed oocysts in their feces. Five negative cats in the Sheather were positive according to qPCR on the 3rd day post-inoculation (dpi). Oocysts were detected on the 4th dpi using the Sheather; however, there was no statistical difference between the two methods (p=0.1116). In addition, there was no statistically significant difference in oocyst shedding between the groups according to the Sheather technique (p=0.6534) and qPCR (p=0.9670). In conclusion, these results demonstrate that qPCR can be used as an alternative to the Sheather to detect and quantify T. gondii oocysts.

Acute infection with Toxoplasma gondii oocysts preferentially activates non-neuronal cells expressing serotonin in the jejunum of rats.

The interaction of Toxoplasma gondii with the gastrointestinal tract of its host is highly regulated. Once ingested, the parasite crosses the epithelium without altering the permeability of the intestinal barrier. Nevertheless, many studies report alterations ranging from structural to functional damage in cells and tissues that make up the wall of the small and large intestine. Although the immune response to the parasite has been extensively studied, the role of serotonin (5-HT) in toxoplasmosis is poorly understood. Here we investigate the distribution of cells expressing 5-HT and its effects on cells and tissues of the jejunal wall of rats after 2, 3, or 7 days of T. gondii infection. KEY RESULTS: Our results show that transposition of the jejunal epithelium by T. gondii leads to ruptures in the basement membrane and activation of the immune system, as confirmed by the decrease in laminin immunostaining and the increase in the number of mast cells, respectively. CONCLUSIONS AND INFERENCES: We showed an increase in the number of enterochromaffin cells and mast cells expressing 5-HT in the jejunal wall. We also observed that the percentage of serotonergic mast cells increased in the total population. Thus, we can suggest that oral infection by T. gondii oocysts preferentially activates non-neuronal cells expressing 5-HT. Together, these results may explain both the changes in the extracellular matrix and the morphology of the enteric ganglia.

Comparison of serological and molecular techniques to detect Toxoplasma gondii in free-range chickens (Gallus gallus domesticus).

The present study aimed to compare different indirect and direct diagnostic techniques to diagnose Toxoplasma gondii in free-range chickens. Samples of 386 chickens obtained from 24 Paraná properties were used for serological analysis by indirect fluorescent antibody test (IFAT), modified agglutination test (MAT), and enzyme-linked immunosorbent assay (ELISA). Animals positive by IFAT and/or MAT had their tissues submitted to the mouse bioassay, and those who were positive in this technique had their blood, tissues, and acidic pepsin tissue digestion submitted to PCR (conventional, nested, and quantitative-PCR (qPCR)). One hundred and nineteen chickens (30.8 %) were positive in at least one of the serological tests, being 102 (26.4 %) in the IFAT, 64 (16.6 %) in the MAT, and 62 (16.0 %) in the ELISA. The IFAT was used as a gold standard, and the MAT showed higher sensitivity (46.0 %) and specificity (94.0) compared to ELISA (43.5 % and 93.6 %, respectively). Ninety samples of eighteen chickens positive in the mouse bioassay were subjected to PCR, and according to molecular tests, the conventional PCR detected the T. gondii DNA in 30 % (27/90) of the samples, in 38.8 % (35/90) with nested-PCR and 40.0 % (36/90) with real-time. According to molecular analyzes, the sensitivity was higher in ITS1 nested-PCR (69.4 %) and specificity in conventional PCR-529bp (90.7 %), using the qPCR as the gold standard. MAT and ELISA had similarities in concordance analyzes. The IFAT was the serological technique with the highest agreement with the mouse bioassay, and serological tests in parallel showed to be a good screening option for the isolation of T. gondii in chick tissues. The PCR markers effectively detected the parasite DNA, and the heart was the tissue with the highest number of positives samples. The conventional PCR had sensitivity similar to nested-PCR and qPCR and could be a cheaper alternative to diagnose T. gondii infection in chicken tissues.

Biogenic silver nanoparticles (AgNp-Bio) reduce Toxoplasma gondii infection and proliferation in HeLa cells, and induce autophagy and death of tachyzoites by apoptosis-like mechanism.

Toxoplasma gondii is a protozoan parasite that can cause severe and debilitating diseases, especially in immunocompromised individuals. The available treatment is based on drugs that have low efficacy, high toxicity, several adverse effects, and need long periods of treatment. Thus, the search for therapeutic alternatives is urgently needed. Biogenic silver nanoparticles (AgNp-Bio) have been associated with several biological effects, as antiproliferative, pro-apoptotic, antioxidant, antiviral, antifungal, and antiprotozoal activity. Thus, the objective was evaluating AgNp-Bio effect on HeLa cells infected with T. gondii (RH strain). First, nontoxic AgNp-Bio concentrations for HeLa cells (1.5 - 6 µM) were determined, which were tested on cells infected with T. gondii. A significant reduction in infection, proliferation, and intracellular parasitic load was observed, also an increase in ROS and IL-6. Additionally, the evaluation of the action mechanisms of the parasite showed that AgNp-Bio acts directly on tachyzoites, inducing depolarization of the mitochondrial membrane, ROS increase, and lipid bodies accumulation, as well as triggering an autophagic process, causing damage to the parasite membrane, and phosphatidylserine exposure. Based on this, it was inferred that AgNp-Bio affects T. gondii by inducing immunomodulation and microbicidal molecules produced by infected cells, and acts on parasites, by inducing autophagy and apoptosis.