Epidemiological studies on toxoplasma gondii infection in a one health perspective

The objective of the present Ph.D. thesis was to investigate with a One Health approach the epidemiological patterns of T. gondii infection in Italy, to better understand the transmission dynamics of the parasite, following different research lines. The results of a retrospective analysis in animals and human showed the widespread distribution of T. gondii in the study area, with specific antibodies found in various animal species and human populations, indicating its constant presence across diverse environments. The environment plays a significant role in T. gondii's epidemiology. Migratory aquatic birds, rodents, wolves, and wild boars were investigated as sentinels of their spread, highlighting the potential transmission across geographic areas and infection risks for wildlife in natural settings. The study also provided insights into seroprevalence in wolves. Dogs, subjected to serological investigations exhibited risk factors for T. gondii infection, such as cohabitation with cats, coprophagy behaviours, and continuous outdoor. Correlation between serological evidence of exposure to T. gondii and pathological anxiety in large-size dogs was observed, and the consumption of raw meat was associated with a higher risk of infection in these animals. Results of the investigations conducted in this thesis, demonstrate the dynamic nature of T. gondii infection in cattle, characterized by new infections and declining antibody levels over the production cycle. The study also describes a co-infection between T. gondii and Sarcocystis hominis in bovine eosinophilic myositis. In the final part of the Thesis, a comprehensive genotyping of T. gondii in Italy reveals the predominance of Type II strains, particularly in cases of ovine abortion and fatal toxoplasmosis among captive Lemur catta. This approach enhances our understanding of the parasite's genetic diversity and transmission patterns, vital for effective management of its impact on human and animal health in Italy.

Occurrence and risk factors associated with infection by Toxoplasma gondii in goats in the State of Alagoas, Brazil

INTRODUCTION: Goats are considered very susceptible to infection by Toxoplasma gondii and when this occurs during pregnancy, it may cause fetal death with subsequent fetal resorption, abortion, mummification, stillborn or the birth of weak goats. The objective of this work was to determine the occurrence of and identify risk factors for T. gondii infection in goats in different mesoregions in the State of Alagoas. METHODS: The research was conducted on 24 goat breeding farms in 10 municipalities. A total of 454 blood samples were examined for anti-T. gondii antibodies by indirect immunofluorescence antibody test. To evaluate the risk factors of toxoplasmosis in goats, questionnaires were applied analyzing the farm's production system and nutritional, reproductive and sanitary management. RESULTS: Disease occurrence was 39% with 95.8% of farms presenting seropositive animals. Significant associations were observed for mesoregion (OR = 0.23; 95%CI = 0.09 - 0.57), age (OR = 0.36; 95%CI = 0.20 - 0.64), semi-intensive herd management (OR = 8.70; 95%CI = 1.87 - 40.43), access of cats to water provided for goats (OR = 3.38; 95%CI = 1.89-6.02) and cats feeding on placental remnants (OR = 2.73; 95%CI = 1.38 - 5.40). CONCLUSIONS: Toxoplasma gondii infection is disseminated in the State of Alagoas and the adoption of a program that monitors infection foci is required to reduce the risk factors identified in the present study.

Effects of infection with Toxoplasma gondii oocysts on the intestinal wall and the myenteric plexus of chicken (Gallus gallus)

This paper aims to analyze the effects of the Toxoplasma gondii infection in the intestinal wall and myenteric plexus of chicken (Gallus gallus). Ten 36-day-old chickens were separated into two groups: control and experimental, orally inoculated with oocysts of the T. gondii strain M7741 genotype III. After 60 days the birds were submitted to euthanasia and had their duodenum removed. Part of the intestinal segments was submitted to histological routine, HE staining, PAS histochemical technique, and Alcian Blue. Qualitative analysis of the intestinal wall and comparative measurements among the groups with respect to total wall thickness, muscle tunic, mucosa, and tunica mucosa were carried out. Caliciform cells were quantified. The other part of the intestinal segments was fixed in formol acetic acid and dissected having the tunica mucosa and the tela submucosa removed. Neurons were stained with Giemsa, counted, and measured. Chickens from the experimental group presented diarrhea and inflammatory infiltrates in the tunica mucosa, thickness reduction of all the parameters assessed in the intestinal wall, and an increase of the number of caliciform cells. There was a ~70% reduction regarding the intensity of myenteric neurons; and the remaining cells presented a reduction of ~2.4% of the perikarion and ~40.5% of the nucleus (p<0.05). Chronic infection induced by T. gondii oocysts resulted in intestinal wall atrophy, mucin secretion increase, death and atrophy of chicken myenteric plexus neurons. Death and atrophy of myenteric plexus neurons may be related with the causes of diarrhea observed in chickens with toxoplasmosis.

SEROPREVALENCE AND ISOLATION OF TOXOPLASMA GONDII FROM SHEEP FROM SAO PAULO STATE, BRAZIL

Sheep are important in the epidemiology of Toxoplasma gondii infection, but little is known of ovine toxoplasmosis in Brazil. Antibodies to T. gondii were assayed in sera of 495 sheep from 36 countries of Sao Paulo state. Brazil, using the modified agglutination test (MAT titer >= 1:25); 120 (24.2%) sheep tested positive. Samples of brain, heart, and diaphragm of 85 seropositive sheep were pooled, digested in pepsin, and bioassayed in mice. Toxoplasma gondii was isolated from tissue homogenated of 16 sheep, and the isolated were designated TgShBr 1-16. Six of the 16 T. gondii isolated killed 100% of infected mice. Results indicate that asymptomatic sheep can harbour mouse-virulent T. gondii; hence, they can serve as a source of infection for humans.

Toxoplasma gondii: The severity of toxoplasmic encephalitis in C57BL/6 mice is associated with increased ALCAM and VCAM-1 expression in the central nervous system and higher blood-brain barrier permeability

In order to investigate the differential ALCAM, ICAM-1 and VCAM-1 adhesion molecules mRNA expression and the blood-brain barrier (BBB) permeability in C57BL/6 and BALB/c mice in Toxoplasma gondii infection, animals were infected with ME-49 strain. It was observed higher ALCAM on day 9 and VCAM-1 expression on days 9 and 14 of infection in the central nervous system (CNS) of C57BL/6 compared to BALB/c mice. The expression of ICAM-1 was high and similar in the CNS of both lineages of infected mice. In addition, C57BL/6 presented higher BBB permeability and higher IFN-gamma and iNOS expression in the CNS compared to BALB/c mice. The CNS of C578L/6 mice presented elevated tissue pathology and parasitism. In conclusion, our data suggest that the higher adhesion molecules expression and higher BBB permeability contributed to the major inflammatory cell infiltration into the CNS of C57BL/6 mice that was not efficient to control the parasite. (C) 2010 Elsevier Inc. All rights reserved.

Prevalence and risk factors for anti-Toxoplasma gondii antibodies in goats of the Serido Oriental microregion, Rio Grande do Norte state, Northeast region of Brazil

The prevalence and risk factors for anti-Toxoplasma gondii antibodies were investigated in goats of the Serido Oriental microregion, Rio Grande do Norte state, Northeast region of Brazil. Three hundred and sixty-six blood samples from goats collected by jugular venopuncture were used. For the serologic diagnosis of Toxoplasma gondii infection, the indirect fluorescent-anti body test (IFAT) with cut-off value 1:64 was carried out. The prevalence of anti-T. gondii antibodies was 30.6% [95% CI = 25.9-35.6%] with titers ranging from 1:64 to 1: 16,384. The multivariate logistic regression analysis showed that the risk factors associated to anti-T. gondii antibodies were presence of cats in the herd, extensive/semi-intensive management systems and lack of mineral supplementation. (c) 2008 Elsevier B.V. All rights reserved.

Avaliação da reatividade de anticorpos anti-Toxoplasma gondii pela técnica de citometria de fluxo como indicadores de lesão ocular em soros de recém-nascidos com toxoplasmose congênita

A retinocoroidite é a manifestação mais comum causada pela infecção congênita por Toxoplasma gondii. Devido a gravidade das lesões oculares que podem até levar à perda completa da visão, a detecção precoce da toxoplasmose congênita e da lesão ocular são essenciais para o tratamento. Este trabalho possuiu o objetivo de avaliar a aplicabilidade da pesquisa de anticorpos IgG e das subclasses IgG1, IgG2, IgG3 e IgG4 anti-T. gondii por citometria de fluxo como marcador laboratorial das diferentes formas de lesões retinocoroidais na toxoplasmose congênita. Foram analisadas 88 amostras de soro de recém-nascidos com toxoplasmose congênita, sendo 25 sem lesão ocular (SL), 10 com lesão ocular ativa (RA), 26 com lesão ocular ativa e cicatricial (RAC) e 27 com lesão ocular cicatricial (RC). Foram também utilizadas 19 amostras de soro de recém-nascidos não infectados que apresentaram IgG positivo após o nascimento (NI). Essas amostras foram obtidas a partir de soros de recémnascidos participantes de um programa de triagem neonatal realizado em Minas Gerais realizado nos anos de 2006 e 2007. Os resultados demonstraram que os recém-nascidos com toxoplasmose congênita apresentam maior reatividade de anticorpos IgG total e subclasses IgG1, IgG2 e IgG3 do que indivíduos não infectados. No grupo não infectado, o único anticorpo com mais de 50% de indivíduos com alta reatividade de anticorpos foi IgG4. Ao comparar os grupos de indivíduos com toxoplasmose congênita, foi observado que o grupo RAC, seguido de RC, apresentou maior reatividade principalmente para os anticorpos IgG1 e IgG3 comparado aos recém-nascidos dos grupos RA e SL, enquanto que pacientes do grupo RA apresentaram maior reatividade para IgG4 do que indivíduos dos outros grupos. IgG1 foi a única subclasse capaz de diferenciar os grupos NI, SL dos grupos RAC e RC. Também foi avaliado o índice de avidez de IgG total, que não permitiu estabelecer nenhum critério de diferenciação das formas de lesão ocular causadas pelo T. gondii. Portanto, a citometria de fluxo demonstrou que pode ser um método laboratorial complementar para ser utilizado como indicador das diferentes lesões oculares causadas pela toxoplasmose congênita.

Prevalence of IgG antibodies specific to Toxoplasma gondii among blood donors in Recife, Northeast Brazil

A serological survey of Toxoplasma gondii infection in blood donors was carried out in order to identify seroprevalence in Recife, Brazil. Sera from 160 individuals (119 male and 41 female) were evaluated by using a Toxoplasma IgG-antibody enzyme immunoassay (Denka Seiken Co., LTD., Tokyo, Japan). The seropositive percentual for males (79.0%) showed to be higher (p < 0.05) than for females (63.4%). This percentage increases with age, ranging from 18.2% to 92.6% for individuals aging under 20 and 40-50 years old, respectively. For women of childbearing age (18-40 years) it was found a prevalence of 51.6%.

Lacrimal secretory IgA in active posterior uveitis induced by Toxoplasma gondii

It is quite difficult to diagnose active toxoplasmosis in patients with ocular toxoplasmosis. Active posterior uveitis presumably due to Toxoplasma gondii infection (APUPT) is seldom produced during a prime-infection; hence most patients do not show high IgM antibodies. High levels of IgA have been described in active toxoplasmosis. The purpose of this study was to investigate possible association between APUPT and the specific anti-parasite sIgA in tears. The study was carried out as case-control. Tears of 25 clinically confirmed APUPT patients and 50 healthy control subjects were analyzed. All were IgG seropositive. Specific sIgA was determined by ELISA assay using T. gondii RH strain crude extract. Anti-T. gondii sIgA was found in 84% of the cases and in 22% of the control subjects. The intensity of the reaction was higher in APUPT cases (P = 0.007). There was strong association between APUPT patients and lacrimal sIgA (odds-ratio 18.61, P = 0.0001). ELISA test sensitivity was 84% and specificity 78% . Our data suggest that anti-T.gondii secretory IgA found in tears may become an important marker for active ocular toxoplasmosis.

Toxoplasma gondii and the Immunity-Related GTPase (IRG) resistance system in mice: a review

The Immunity Related GTPases (IRG proteins) constitute a large family of interferon-inducible proteins that mediate early resistance to Toxoplasma gondii infection in mice. At least six members of this family are required for resistance of mice to virulent T. gondii strains. Recent results have shown that the complexity of the resistance arises from complex regulatory interactions between different family members. The mode of action against T. gondii depends on the ability of IRG proteins to accumulate on the parasitophorous vacuole of invading tachyzoites and to induce local damage to the vacuole resulting in disruption of the vacuolar membrane. Virulent strains of T. gondiiovercome the IRG resistance system, probably by interfering with the loading of IRG proteins onto the parasitophorous vacuole membrane. It may be assumed that T. gondii strains highly virulent for mice will be disadvantaged in the wild due to the rapid extinction of the infected host, while it is self-evident that susceptibility to virulent strains is disadvantageous to the mouse host. We consider the possibility that this double disadvantage is compensated in wild populations by segregating alleles with different resistance and susceptibility properties in the IRG system.

Vaccines against Toxoplasma gondii: challenges and opportunities

Development of vaccines against Toxoplasma gondii infection in humans is of high priority, given the high burden of disease in some areas of the world like South America, and the lack of effective drugs with few adverse effects. Rodent models have been used in research on vaccines against T. gondii over the past decades. However, regardless of the vaccine construct, the vaccines have not been able to induce protective immunity when the organism is challenged with T. gondii, either directly or via a vector. Only a few live, attenuated T. gondii strains used for immunization have been able to confer protective immunity, which is measured by a lack of tissue cysts after challenge. Furthermore, challenge with low virulence strains, especially strains with genotype II, will probably be insufficient to provide protection against the more virulent T. gondii strains, such as those with genotypes I or II, or those genotypes from South America not belonging to genotype I, II or III. Future studies should use animal models besides rodents, and challenges should be performed with at least one genotype II T. gondii and one of the more virulent genotypes. Endpoints like maternal-foetal transmission and prevention of eye disease are important in addition to the traditional endpoint of survival or reduction in numbers of brain cysts after challenge.

Culture of mouse peritoneal macrophages with mouse serum induces lipid bodies that associate with the parasitophorous vacuole and decrease their microbicidal capacity against Toxoplasma gondii

Lipid bodies [lipid droplets (LBs)] are lipid-rich organelles involved in lipid metabolism, signalling and inflammation. Recent findings suggest a role for LBs in host response to infection; however, the potential functions of this organelle in Toxoplasma gondii infection and how it alters macrophage microbicidal capacity during infection are not well understood. Here, we investigated the role of host LBs in T. gondii infection in mouse peritoneal macrophages in vitro. Macrophages cultured with mouse serum (MS) had higher numbers of LBs than those cultured in foetal bovine serum and can function as a model to study the role of LBs during intracellular pathogen infection. LBs were found in association with the parasitophorous vacuole, suggesting that T. gondii may benefit from this lipid source. Moreover, increased numbers of macrophage LBs correlated with high prostaglandin E2 (PGE2) production and decreased nitric oxide (NO) synthesis. Accordingly, LB-enriched macrophages cultured with MS were less efficient at controlling T. gondii growth. Treatment of macrophages cultured with MS with indomethacin, an inhibitor of PGE2 production, increased the microbicidal capacity against T. gondii. Collectively, these results suggest that culture with MS caused a decrease in microbicidal activity of macrophages against T. gondii by increasing PGE2 while lowering NO production.

Sparrows (Passer domesticus L.) as intermediary hosts of Toxoplasma gondii in poultry farms from the "agreste" region of Pernambuco, Brazil

This paper aimed to identify Toxoplasma gondii infection in house sparrows (Passer domesticus, Linneaus 1758) coming from poultry farms in the "agreste" region of the Brazilian state of Pernambuco. 151 sparrows (Passer domesticus) captured in eight broiler, egg layer and commercial laying poultry farms, were used. Indirect hemagglutination test was used to research anti-T. gondii antibodies. Animals that presented titration of 1:16 were destined to DNA research through Polymerase Chain Reaction (PCR) technique, followed by Nested-PCR. It was observed that, from 151 analyzed samples. 91 (60.3%) were reagents and 60 (39.7%) were not reagents. It was verified, through analysis of the distribution of infected animals frequency per farm, that in only one farm (12.5%) no animal reagent to T. gondii was captured. It was also observed that three (30.00%) of the ten samples destined to DNA research for T. gondii were positive to PCR and four (40.00%) were positive to Nested-PCR. Anti-T gondii antibodies occurrence and the molecular identification of the agent confirmed natural T. gondii infection in sparrows from poultry farms in Brazil. Other studies must be carried out to highlight the real importance of these animals in the epidemiological chain and their efficiency in the transmission of the parasite to felines. Therefore, researches that use parasite isolation and molecular techniques to determine genomic profile of the agent present in these poultry farms are needed.

First isolation and molecular characterization of Toxoplasma gondii from finishing pigs from São Paulo State, Brazil

Toxoplasma gondii infection is widely prevalent in humans in Brazil. Among the food animals, pigs are considered the most important meat source of T. gondii for infection in humans. In the present study, we report the first isolation of viable T. gondii from finishing pigs in Brazil. Antibodies to T. gondii were found in 49 (17%) of 286 pigs prior slaughter using the modified agglutination test (MAT) at a serum dilution of 1:25. Attempts were made to isolate T. gondii from 28 seropositive pigs. Samples of heart, brain, and tongue from each pig were pooled, digested in acid pepsin, and bioassayed in five mice per pig. Viable T. gondii was isolated from seven pigs; all isolates were lethal for mice. Restriction fragment length polymorphism on products of SAG2 locus amplified by PCR revealed that two isolates were Type I and five were Type III. The results indicate that phenotypically and genetically T. gondii isolates from pigs from Brazil are distinct from isolates of T gondii from pigs in the USA. (c) 2005 Elsevier B.V. All rights reserved.

Histopathological analysis of the reproductive system of male dogs experimentally infected with Toxoplasma gondii

O presente trabalho teve como objetivo descrever eventuais alterações histopatológicas no sistema reprodutor (testículo e epidídimo) de cães machos experimentalmente infectados com Toxoplasma gondii. Para tal, 10 animais sorologicamente negativos para T. gondii foram selecionados e distribuídos em três grupos experimentais: GI - três cães inoculados com 2,0 x 10(5) oocistos da cepa P, GII - três cães infectados com 1,0 x 10(6) taquizoítos da cepa RH e GIII - quatro cães mantidos como controle. Pesquisa de anticorpos (IFI) contra T. gondii foi realizada. A infecção por T. gondii confirmou-se pela soroconversão de todos os machos infectados a partir do 7° e do 14° dia pós-inoculação (DPI) para cães que receberam taquizoítos e oocistos respectivamente. Decorridos 70DPI, realizou-se, em todos os cães, orquiectomia, e amostras (testículo e epidídimo) foram coletadas e processadas histologicamente para leitura em microscópio óptico. As seguintes alterações foram diagnosticadas: infiltrado inflamatório mononuclear leve e moderado em epidídimo, edema celular moderado, degeneração hidrópica e fibrose intersticial moderada em túbulos seminíferos. Os resultados histopatológicos do presente trabalho, aliados ao isolamento do T. gondii em fragmentos de testículo e epidídimo pela imunoistoquímica, juntamente com os resultados encontrados na literatura por outros autores em diferentes tecidos, permitem inferir que as alterações encontradas nos cães infectados com o respectivo protozoário são sugestivas de infecção toxoplásmica.