Use of near-infrared Raman spectroscopy to detect IgG and IgM antibodies against Toxoplasma gondii in serum samples of domestic cats

Near-infrared Raman spectroscopy (NIRS) is a particularly promising technique that is being used in recent years for many biomedical applications. Optical spectroscopy has gained increasing prominence as a tool for quantitative analysis of biological samples, clinical diagnostic, concentration measurements of blood metabolites and therapeutic drugs, and analysis of the chemical composition of human tissues. Toxoplasmosis is an important zoonosis in public health, and domestic cats are the most important transmitters of the disease. This disease can be detected by several serological tests, which usually have a high cost and require a long time. The goal of this work was to investigate a new method to diagnosis Toxoplasma gondii infections using NIRS. In order to confirm antibody detection, 24 cat blood scrum samples were analyzed by the Raman spectra, from which 23 presented positive serology to toxoplasmosis and one was a reference negative serum. Characteristic Raman peaks allowed differentiation between negative and positive sera, confirming the possibility of antibody detection by Raman spectroscopy. These results give the first evidence that this technique can be useful to quantify antibodies in cat sera.

Seroepidemiology of Toxoplasma gondii, Neospora caninum, and Leishmania spp. infections and risk factors for cats from Brazil

The seroprevalence of infection by Toxoplasma gondii, Neospora caninum, and Leishmania spp. was detected through an indirect immunofluorescence in 70 cats from the Andradina Municipality, São Paulo State, Brazil. Anti-T. gondii antibodies (titer >64) were detected in 15.7% (11/70) of animals, whereas positivity for N. caninum (titer 16) was not observed in any animal. of the cats from urban and rural areas, 10.4% (5/48) and 27.2% (6/22) were positive for T. gondii, respectively. Breed, age, food, and contact with animals of other species were significant for considering the positivity for T. gondii (P <= 0.0001). Cats having access to streets (17.1%, 11/64), cats cohabiting with rats (19.6%, 10/51), and cats feeding on homemade food and raw milk (27.2%, 6/22) were positive for T. gondii. In addition, 4.2% (3/70) of the cats were positive for Leishmania spp. by ELISA technique and negative by IFAT without coinfection with T. gondii and Leishmania spp. There was no serological positivity against feline immunodeficiency virus or feline leukemia virus. In conclusion, T. gondii infection in part of the feline population from Andradina is not linked to immunosuppressions or coinfections but probably to postnatal infection in association with the type of diet and presence of rats.

Serotyping of Toxoplasma gondii in cats (Felis domesticus) reveals predominance of type II infections in Germany.

BACKGROUND Cats are definitive hosts of Toxoplasma gondii and play an essential role in the epidemiology of this parasite. The study aims at clarifying whether cats are able to develop specific antibodies against different clonal types of T. gondii and to determine by serotyping the T. gondii clonal types prevailing in cats as intermediate hosts in Germany. METHODOLOGY To establish a peptide-microarray serotyping test, we identified 24 suitable peptides using serological T. gondii positive (n=21) and negative cat sera (n=52). To determine the clonal type-specific antibody response of cats in Germany, 86 field sera from T. gondii seropositive naturally infected cats were tested. In addition, we analyzed the antibody response in cats experimentally infected with non-canonical T. gondii types (n=7). FINDINGS Positive cat reference sera reacted predominantly with peptides harbouring amino acid sequences specific for the clonal T. gondii type the cats were infected with. When the array was applied to field sera from Germany, 98.8% (85/86) of naturally-infected cats recognized similar peptide patterns as T. gondii type II reference sera and showed the strongest reaction intensities with clonal type II-specific peptides. In addition, naturally infected cats recognized type II-specific peptides significantly more frequently than peptides of other type-specificities. Cats infected with non-canonical types showed the strongest reactivity with peptides presenting amino-acid sequences specific for both, type I and type III. CONCLUSIONS Cats are able to mount a clonal type-specific antibody response against T. gondii. Serotyping revealed for most seropositive field sera patterns resembling those observed after clonal type II-T. gondii infection. This finding is in accord with our previous results on the occurrence of T. gondii clonal types in oocysts shed by cats in Germany.

Estudo do metabolismo mitocondrial e da resposta anti-apoptótica de células endoteliais humanas durante a evolução da infecção por taquizoítos de Toxoplasma gondii

A toxoplasmose é uma zoonose amplamente distribuída que afeta mais de um terço da população mundial e de grande importância na saúde pública. A maioria das infecções em humanos por Toxoplasma gondii é assintomática. A toxoplasmose é amplamente investigada visto que se apresenta como uma doença grave em pessoas imunodeprimidas (portadores da síndrome da imunodeficiência adquirida (SIDA), não tratados, indivíduos transplantados, paciente em tratamento quimioterápico ou em uso de drogas supressoras e gestantes). A toxoplasmose congênita frequentemente pode levar ao aborto espontâneo ou até mesmo resultar na formação de crianças com algum grau de atraso no desenvolvimento mental e/ou físicos, deste modo, a transmissão congênita pode ser muito mais importante do que se pensava, pois os parasitos encontrados na circulação sanguinea são capazes de infectar as células endoteliais dos vasos e os tecidos circunjacentes, podendo resultar no encistamento do T. gondii. Atualmente a toxoplasmose vem sendo investigada devido a sua associação a inúmeras outras doenças, assim, estudos sobre a evolução da infecção por T. gondii em diferentes tipos de células hospedeiras se fazem necessários para uma abordagem terapêutica adequada. Ao invadir a célula hospedeira o parasito possui a capacidade de recrutar as mitocôndrias promovendo mudanças na organização mitocondrial ao longo da progressão da infecção, garantindo um ambiente favorável a sua multiplicação. Diante disso, investigamos se o parasito possui a capacidade de interferir no metabolismo mitocondrial e na resposta apoptótica da célula endotelial. O presente trabalho teve como objetivo analisar o metabolismo mitocondrial através da respirometria de alta-resolução e da resposta apoptótica através do western blotting das células endoteliais da veia umbilical humana (HUVEC) infectadas por 2, 6 e 20 horas por taquizoítos de T. gondii. A respirometria de alta-resolução revelou que o parasito interfere no metabolismo energético da célula hospedeira. A análise do conteúdo de proteínas da família Bcl-2 por western blotting revelou maior estímulo apoptótico no tempo inicial de infecção, quando comparado aos demais tempos. Os resultados dos conteúdos de caspase 3, proteína efetora da apoptose, não demonstrou diferença nos tempos iniciais de infecção Entretanto, em tempos mais tardios, o conteúdo de caspase 3 mostrou-se significativamente aumentado quando comparado às HUVEC não infectadas. A dinâmica de replicação do parasito foi observada através do monitoramento pelo sistema Time-Lapse Nikon BioStation IMQ em tempo real das células infectadas por T.gondii. Portanto, nossos resultados sugerem que o protozoário ao recrutar as mitocôndrias da célula hospedeira interfere no metabolismo mitocondrial e na modulação da apoptose para garantir um ambiente favorável a sua multiplicação.

Immunological Interactions between 2 Common Pathogens, Th1-Inducing Protozoan Toxoplasma gondii and the Th2-Inducing Helminth Fasciola hepatica

Background: The nature of the immune response to infection is dependent on the type of infecting organism. Intracellular organisms such as Toxoplasma gondii stimulate a Th1-driven response associated with production of IL-12, IFN-gamma, nitric oxide and IgG2a antibodies and classical activation of macrophages. In contrast, extracellular helminths such as Fasciola hepatica induce Th2 responses characterised by the production of IL-4, IL-5, IL-10 and IgG1 antibodies and alternative activation of macrophages. As co-infections with these types of parasites commonly exist in the field it is relevant to examine how the various facets of the immune responses induced by each may influence or counter-regulate that of the other.

Leptospira spp. and Toxoplasma gondii antibodies in vampire bats (Desmodus rotundus) in Botucatu region, SP, Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Detection and molecular analysis of Toxoplasma gondii and Neospora caninum from dogs with neurological disorders

Introduction: Toxoplasma gondii and Neospora caninum are related Apicomplexa parasites responsible for systemic diseases in many species of animals, including dogs. Methods: This study aimed to determine the occurrence of T. gondii and N. caninum infections in 50 dogs with neurological signs that were admitted to the Veterinary Hospital of Universidade Estadual Paulista, City of Botucatu, Brazil. All animals were screened for antibodies using an immunofluorescent antibody test for both parasites. Tissues of positive animals were bioassayed in mice (T. gondii) and gerbils (N. caninum), and DNA was analyzed using the polymerase chain reaction (PCR). Positive samples for T. gondii by PCR were typed using restriction fragment length polymorphism-PCR for 11 markers: SAG1, SAG2 (5'-3'-SAG2 and alt.SAG2), SAG3, Btub, GRA6, L358, c22-8, c29-6, PK1 and Apico, and CS3 marker for virulence analysis. Results: Specific antibodies were detected in 11/50 (22%; 95% confidence interval (CI95%), 12.8-35.3%) animals for T. gondii and 7/50 (14%; CI95%, 7.02-26.3%) for N. caninum. In the bioassay and PCR, 7/11 (63.6%; CI95%, 34.9-84.8%) samples were positive for T. gondii and 3/7 (42.9%; CI95% I, 15.7-75.5%) samples were positive for N. caninum. Three different genotypes were identified, but only 1 was unique. Conclusions: These data confirm the presence of T. gondii and N. caninum in dogs from Brazil, indicating the importance of this host as a sentinel of T. gondii for human beings, and the genotypic variation of this parasite in Brazil.

Serological profile of Toxoplasma gondii and Neospora caninum infection in commercial sheep from São Paulo State, Brazil

Neosporosis and toxoplasmosis are two important infections in young and adult sheep, leading to low production and abortion. This study aimed to determine the frequency of antibodies to Toxoplasma gondii and Neospora caninum in sheep from the eastern region of São Paulo State, Brazil. Serum samples (382) were collected from the sheep and assayed for T. gondii through modified agglutination test (MAT) and indirect fluorescent antibody test (IFAT), and for N. caninum antibodies, through IFAT, with cut-off titers equal to 16 (T. gondii) and 25 (N. caninum). All frozen samples were sent to the Center for Zoonoses Research (NUPEZO), Department of Veterinary Hygiene and Public Health (DHSVP), FMVZ, UNESP, for serological tests. A total of 71/382(18.6%) samples reacted to T. gondii, especially at titers 16 (28; 39.4%), 64(15; 21.1%), 256 (21; 29.6%) and 1024 (6; 8.5%) by MAT, and 16(34: 47.9%), 64 (18; 25.4%), 256 (14; 19.7%) and 1024 (5; 7%) by IFAT. As regards N. caninum, 49/382 (12.8%) samples reacted at titers 25 (17; 34.7%), 50 (11; 22.5%), 100 (11; 22.5%), and >= 200 (10; 20.4%). These animals presented infection but no clinical signs. Six and ten animals had high titers for toxoplasmosis and neosporosis. No significant association was observed between antibodies for both parasites (P = 0.535) according to Fisher's exact test, and no correlation was found between T. gondii (MAT) and N. caninum antibody titers (r = -0.0068; P=0.895), T. gondii (IFAT) and N. caninum antibody titers (r = -0.0025; P = 0.961). Thus, T. gondii and N. caninum infections were observed in farms located in São Paulo State, where sheep play an important economical role for the national and regional business. (C) 2010 Elsevier By. All rights reserved.

Seroprevalence of Toxoplasma gondii and Neospora caninum in captive maned wolves (Chrysocyon brachyurus) from southeastern and midwestern regions of Brazil

The main purpose of the present study was to investigate the occurrence of antibodies against T gondii and N. caninum in captive maned wolves from Brazil, considering that little information is available at the literature about infections by these parasites in this wild animal. Serum samples were obtained from 59 maned wolves originated from six zoos and from one ecological reserve of the southeastern and midwestern regions of Brazil. To detect IgG antibodies against T gondii, an ELISA protocol was used and the results were expressed as ELISA reactivity indexes (131). Serology for N. caninum was carried out by indirect fluorescent antibody test (IFAT) and cut-off titers were established at 1:25 dilution. From the total of the analyzed samples, 44 (74.6%) were seropositive for T gondii and only 5 (8.5%) for N. caninum. Seropositivity for T gondii ranged from 0 to 100% in the seven different origin locals, with rates over 50% among the six zoos, whereas no positivity was found in the samples from ecological reserve. For N. caninum, seroprevalence varied from 0 to 50% in the different locals, with the highest rates also detected in zoos. Seroprevalence for T gondii was strongly related with age, with rates significantly higher among adult wolves (91.7%) when compared to newborn or young animals. Seropositive samples for N. caninum were found predominantly in adult wolves. For both parasites, seroprevalence did not show a significant distinction in relation to gender. Although seroprevalence for T gondii was significantly higher when compared to N. caninum in the Brazilian captive maned wolves tested, these findings reflect the great exposure of this species to T gondii and, in lower extension, to N. caninum. Also, the present study demonstrated for the first time the presence of antibodies to N. caninum in wild life from South America. (C) 2004 Elsevier B.V. All rights reserved.

Mast cells in the eyes of Calomys callosus (Rodentia : Cricetidae) infected by Toxoplasma gondii

The mast cell is a powerful effector cell for the innate immune system, acting through the secretion of several distinct mediators. Few studies have demonstrated the relationship between mast cells and toxoplasmosis. In this study, mast cells were investigated in two experimental Toxoplasma infections using Calomys callosus (Rodentia: Cricetidae) as the host. Animals were inoculated either intraperitoneally or via the conjunctiva with tachyzoites of Toxoplasma gondii (RH strain) and sacrificed after 5 days or 24 h, respectively. Enucleated eyes were processed for histological and ultrastructural analysis. Neither experimental infection altered the localization of mast cells compared to control eyes, but they did lead to an accumulation in some tissues as well as to their activation. There was a significant increase in the number of mast cells within 5 days and 24 h after infection. The ocular lesions were characterized by the presence of tachyzoites, inflammatory cells and vasodilatation in the iris and retina. In conclusion, mast cells were mobilized in these experimental infections, suggesting that they play an important role in the host inflammatory response after infection with T. gondii.

Cytomegalovirus and Toxoplasma gondii seroprevalence in a Brazilian liver transplant waiting list

Cytomegalovirus (CMV) disease is a major cause of morbidity and mortality in solid organ transplantation. Disseminated toxoplasmosis after liver transplantation is a rare but fatal event. Serologic screening of the donor and the recipient is essential to prophylactic management, early diagnosis and therapeutic strategies to minimize the consequences of these infections. The aim of the present study was to determine the seroprevalence of CMV and Toxoplasma gondii (TG) in a Brazilian liver transplant waiting list (LTWL). Serological data were collected from 44 candidates on the LTWL between May 2003 and November 2004. Serological investigation of antibodies IgM and IgG against CMV (anti-CMV) and TG (anti-T. gondii) was performed using fluorometry commercial kits. IgG anti-CMV was positive in 37 patients (94.9%) out of 39 available results. There were not IgM anti-CMV positive results. Out of 36 analyzed patients, 22 (61.1%) presented positive IgG anti-T. gondii and none had positive IgM anti-T. gondii. The high CMV seroprevalence among our LTWL reinforces the need for appropriate protocols to avoid related complications, like reactivation and superinfection by CMV. Environmental and drug prophylactic strategies against primary infection and reactivation, as well as early diagnosis and treatment of toxoplasmosis complications, are essential for the good outcome of transplant patients.

Detecção de anticorpos anti-Toxoplasma gondii em cavalos carroceiros da região metropolitana de Curitiba, Paraná, Brasil

Toxoplasma gondii, the agent for toxoplasmosis, has worldwide distribution. Horses normally play a secondary role in its life cycle, but movement around urban areas, feeding on grass and the increasing use of carthorses for gathering recyclable material in some urban areas of Brazil may increase their exposure to T. gondii infection. The aim of the present study was to investigate the frequency of anti-T. gondii antibodies in carthorses in the metropolitan region of Curitiba, PR. IgG antibodies against T. gondii were detected using the indirect fluorescence antibody test (IFAT) (titers ≥ 64). Seventeen (17.0%) of the 100 horses sampled were seropositive. There were no statistical differences in relation to sex (p = 0.28) or age (p = 0.15). Our findings suggest that carthorses are exposed to T. gondii infections and that no associations with age or sex exist.

Falhas reprodutivas associadas com a presença de do vírus da artrite-encefalite caprina, toxoplasma gondii e neospora caninum em caprinos no estado de São Paulo, Brasil

This study aimed at assessing the occurrence of antibodies against the caprine arthritis-encephalitis virus (CAEV), Toxoplasma gondii and Neospora caninum, as well as the associations between the presence of antibodies and the occurrence of reproductive failures in goats. Serum samples were collected from 923 goats of both sexes, over 3 months of age, from 17 dairy farms located in different municipalities of São Paulo State, Brazil. Infections by T. gondii, N. caninum and CAEV were evaluated by indirect methods of diagnosis based on indirect fluorescence antibody test (IFAT), Neospora agglutination test (NAT), and agar gel immunodiffusion (AGID), respectively. A survey was conducted on the farms to obtain information about reproduction dates (abortions, stillbirths and births of weak and premature kids) and zoosanitary management. Antibodies against CAEV, T. gondii and N. caninum was found in 37.81%, 23.62% and 17.23% respectively. There was no significant association between the presence of anti-CAEV antibodies and CAEV/T. gondii or CAEV/N. caninum co-infection, suggesting that CAEV does not predispose goats to infection by these agents. However, when CAEV/T. gondii (p<0.01) or CAEV/N. caninum (p<0.001) co-infection was present, the occurrence of reproductive failures was significantly higher what could indicate that CAEV-induced immunosuppression may predispose goats to develop the clinical symptoms of toxoplasmosis and neosporosis increasing the risks of the reproductive failures.

T and B lymphocytes in the brains of dogs with concomitant seropositivity to three pathogenic protozoans: Leishmania chagasi, Toxoplasma gondii and Neospora caninum

Background: Visceral leishmaniasis is a disease with great variability regarding the clinical manifestations in humans and dogs. Chronically infected dogs may develop neurological disorders, however, there are few reports that characterize the lesions and make clear the pathogenesis of the canine cerebral leishmaniasis. Concomitant with Leishmania chagasi, dogs may be infected by opportunistic pathogens, such as Toxoplasma gondii and Neospora caninum, which may contribute to the occurrence of lesions in the central nervous system. Hence, we aimed to compare the T and B lymphocytes population in the brains of infected dogs with seropositivity to L. chagasi, T. gondii and N. caninum concurrently (n = 24), seropositivity only to L. chagasi (n = 31), and seropositivity to T. gondii and N. caninum (n = 16). Uninfected dogs were used as control (n = 10). Results: Inflammatory lesions, characterised by mononuclear cell accumulation, composed mainly of CD3+ T lymphocytes predominated in several encephalic regions of the dogs from all the three infected groups, with no difference among them (P = 0.0004), whereas CD79α+ B lymphocytes were detected in very small intensity and presented no difference among groups (P = 0.5313). Furthermore, no association among diseases was detected at the serological enquire. Conclusions: We demonstrate that the peripheral infection by L. chagasi per se can promote the influx of lymphocytes within the nervous milieu as occurs during Toxoplasma and Neospora infections, and the concomitant seropositivity against these pathogens does not exacerbate the inflammatory brain lesions. Therefore, these findings give additional support that the brain should be included in the list of organs affected by visceral leishmaniasis and that even asymptomatic infected dogs may develop brain lesions. © 2013 Sakamoto et al.; licensee BioMed Central Ltd.

Sistema histo-sangüíneo ABO, status Secretor e anticorpos anti-Toxoplasma gondii em gestação da região Noroeste do Estado de São Paulo: um estudo de associação

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)