Evidence of SARS-CoV-2 infection and co-infections in stray cats in Brazil.

The zoonotic virus SARS-CoV-2, which causes severe acute respiratory syndrome in humans (COVID-19), has been identified in cats. Notably, most positive cases were in cats that had close contact with infected humans, suggesting a role for humans in animal transmission routes. Previous studies have suggested that animals with immune depletion are more susceptible to SARS-CoV-2 infection. To date, there is limited evidence of SARS-CoV-2 infections in stray and free-range cats affected by other pathogens. In this study, we investigated infections caused by SARS-CoV-2, Leishmania spp., Toxoplasma gondii, Mycoplasma spp., Bartonella spp., Feline leukemia virus (FeLV), and Feline immunodeficiency virus (FIV) in stray cats from an urban park in Brazil during the COVID-19 pandemic. From February to September 2021, 78 mixed-breed cats were tested for SARS-CoV-2 and hemopathogens using molecular analysis at Américo Renné Giannetti Municipal Park, Belo Horizonte, Minas Gerais, Brazil. An enzyme-linked immunosorbent assay (ELISA) was used to detect IgG in T. gondii. None of the animals in this study showed any clinical signs of infections. The SARS-CoV-2 virus RNA was detected in 7.7 % of cats, and a whole virus genome sequence analysis revealed the SARS-CoV-2 Delta lineage (B.1.617.2). Phylogenetic analysis showed that SARS-CoV-2 isolated from cats was grouped into the sublineage AY.99.2, which matches the epidemiological scenario of COVID-19 in the urban area of our study. Leishmania infantum was detected and sequenced in 9 % of cats. The seroprevalence of T. gondii was 23.1 %. Hemotropic Mycoplasma spp. was detected in 7.7 % of the cats, with Mycoplasma haemofelis and Candidatus Mycoplasma haemominutum being the most common. Bartonella henselae and Bartonella clarridgeiae were detected in 38.5 % of the cats, FeLV was detected in 17,9 %, and none of the cats studied tested positive for FIV. This study reports, for the first time, the SARS-CoV-2 infection with whole-genome sequencing in stray cats in southeastern Brazil and co-infection with other pathogens, including Bartonella spp. and Feline leukemia virus. Our study observed no correlation between SARS-CoV-2 and the other detected pathogens. Our results emphasize the importance of monitoring SARS-CoV-2 in stray cats to characterize their epidemiological role in SARS-CoV-2 infection and reinforce the importance of zoonotic disease surveillance.

Chronic infection by atypical Toxoplasma gondii strain induces disturbance in microglia population and altered behaviour in mice.

Toxoplasma gondii chronic infection is characterized by the establishment of tissue cysts in the brain and increased levels of IFN-γ, which can lead to brain circuitry interference and consequently abnormal behaviour in mice. In this sense, the study presented here sought to investigate the impact of chronic infection by two T. gondii strains in the brain of infection-resistant mice, as a model for studying the involvement of chronic neuroinflammation with the development of behavioural alterations. For that, male BALB/c mice were divided into three groups: non-infected (Ni), infected with T. gondii ME49 clonal strain (ME49), and infected with TgCkBrRN2 atypical strain (CK2). Mice were monitored for 60 days to establish the chronic infection and then submitted to behavioural assessment. The enzyme-linked immunosorbent assay was used for measurement of specific IgG in the blood and levels of inflammatory cytokines and neurotrophic factors in the brain, and the cell's immunophenotype was determined by multiparametric flow cytometry. Mice infected with ME49 clonal strain displayed hyperlocomotor activity and memory deficit, although no signs of depressive- and/or anxiety-like behaviour were detected; on the other hand, chronic infection with CK2 atypical strain induced anxiety- and depressive-like behaviour. During chronic infection by CK2 atypical strain, mice displayed a higher number of T. gondii brain tissue cysts and inflammatory infiltrate, composed mainly of CD3+ T lymphocytes and Ly6Chi inflammatory monocytes, compared to mice infected with the ME49 clonal strain. Infected mice presented a marked decrease of microglia population compared to non-infected group. Chronic infection with CK2 strain produced elevated levels of IFN-γ and TNF-ɑ in the brain, decreased NGF levels in the prefrontal cortex and striatum, and altered levels of fractalkine (CX3CL1) in the prefrontal cortex and hippocampus. The persistent inflammation and the disturbance in the cerebral homeostasis may contribute to altered behaviour in mice, as the levels of IFN-γ were shown to be correlated with the behavioural parameters assessed here. Considering the high incidence and life-long persistence of T. gondii infection, this approach can be considered a suitable model for studying the impact of chronic infections in the brain and how it impacts in behavioural responses.

Molecular detection of Toxoplasma gondii in meat for human consumption in Ibague, Colombia / Detección molecular de Toxoplasma gondii en carnes para consumo humano en Ibagué, Colombia

Introduction: Toxoplasma gondii is a parasite with great zoonotic potential. It can infect a broad range of warm-blooded hosts (including livestock) and causes significant losses in the industry. In humans, it has been described as a pathogen in immunosuppressed people, it affects the fetus development in congenital infections, and is associated with various behavioral disorders in healthy people. Humans can acquire T. gondii by consuming undercooked, contaminated meat. Objective: To determine T. gondii positivity (currently unknown) in meat for human consumption (i.e., beef, chicken, and pork) in the city of Ibague, Colombia. Materials and methods: We used conventional nested PCR and the T. gondii B1 gene sequence as amplification target. We collected samples of meat (N=186) sold in the urban area of Ibagué (62 beef, 62 chicken, and 62 pork samples) and determined the T. gondii positivity percentage for each type of meat. Results: The study found an average of 18.8% positivity for all meat samples, pork having the highest percentage (22.5%; 14/62), followed by beef (19.3%; 12/62) and chicken (14.5%; 9/62). The best-amplified products were sequenced by macrogen and aligned with the B1 gene sequences in GenBank, thereby confirming their identity. Conclusions: This is the first study of T. gondii prevalence in meat for human consumption carried out in the city of Ibagué and the department of Tolima. All three types of meat sampled represent a risk for local human infection

Introducción. Toxoplasma gondii es un parásito con gran potencial zoonótico que puede infectar un amplio rango de huéspedes de sangre caliente, incluidos los animales del sector pecuario, lo que causa pérdidas a la industria. En el humano, es patógeno en personas inmunosuprimidas y afecta el desarrollo del feto en infecciones congénitas. Además, se asocia con diversos trastornos del comportamiento en personas sanas. El humano puede adquirir T. gondii al consumir carnes contaminadas mal cocidas. Objetivo. Determinar la positividad de T. gondii en carnes de consumo humano (res, pollo y cerdo) en Ibagué, Colombia. Materiales y métodos. Se utilizó la PCR convencional anidada y la secuencia del gen B1 de T. gondii como blanco de amplificación. Se tomaron 186 muestras de carne comercializada en la zona urbana de Ibagué (62 de res, 62 de pollo y 62 de cerdo) y se obtuvo el porcentaje de positividad en cada tipo de carne evaluada. Resultados. Se encontró un porcentaje de positividad de 18,8 % en las muestras, siendo la carne de cerdo la del mayor porcentaje (22,5 %; 14/62), seguida por las muestras de carne de res (19,3 %; 12/62) y de pollo (14,5 %; 9/62). Los mejores productos amplificados fueron secuenciados en Macrogen, y alineados con las secuencias del gen B1 depositadas en el GenBank, con lo que se confirmó su identidad. Conclusiones. Este es el primer estudio sobre prevalencia de T. gondii en carnes para consumo humano en Ibagué y el departamento del Tolima. Se demostró que los tres tipos de carne representan un riesgo para la infección en humanos a nivel local.

Detección molecular de Toxoplasma gondii en carnes para consumo humano en Ibagué, Colombia / Molecular detection of Toxoplasma gondii in meat for human consumption in Ibague, Colombia

Introducción. Toxoplasma gondii es un parásito con gran potencial zoonótico que puede infectar un amplio rango de huéspedes de sangre caliente, incluidos los animales del sector pecuario, lo que causa pérdidas a la industria. En el humano, es patógeno en personas inmunosuprimidas y afecta el desarrollo del feto en infecciones congénitas. Además, se asocia con diversos trastornos del comportamiento en personas sanas. El humano puede adquirir T. gondii al consumir carnes contaminadas mal cocidas. Objetivo. Determinar la positividad de T. gondii en carnes de consumo humano (res, pollo y cerdo) en Ibagué, Colombia. Materiales y métodos. Se utilizó la PCR convencional anidada y la secuencia del gen B1 de T. gondii como blanco de amplificación. Se tomaron 186 muestras de carne comercializada en la zona urbana de Ibagué (62 de res, 62 de pollo y 62 de cerdo) y se obtuvo el porcentaje de positividad en cada tipo de carne evaluada. Resultados. Se encontró un porcentaje de positividad de 18,8 % en las muestras, siendo la carne de cerdo la del mayor porcentaje (22,5 %; 14/62), seguida por las muestras de carne de res (19,3 %; 12/62) y de pollo (14,5 %; 9/62). Los mejores productos amplificados fueron secuenciados en Macrogen, y alineados con las secuencias del gen B1 depositadas en el GenBank, con lo que se confirmó su identidad. Conclusiones. Este es el primer estudio sobre prevalencia de T. gondii en carnes para consumo humano en Ibagué y el departamento del Tolima. Se demostró que los tres tipos de carne representan un riesgo para la infección en humanos a nivel local.

Introduction: Toxoplasma gondii is a parasite with great zoonotic potential. It can infect a broad range of warm-blooded hosts (including livestock) and causes significant losses in the industry. In humans, it has been described as a pathogen in immunosuppressed people, it affects the fetus development in congenital infections, and is associated with various behavioral disorders in healthy people. Humans can acquire T. gondii by consuming undercooked, contaminated meat. Objective: To determine T. gondii positivity (currently unknown) in meat for human consumption (i.e., beef, chicken, and pork) in the city of Ibague, Colombia. Materials and methods: We used conventional nested PCR and the T. gondii B1 gene sequence as amplification target. We collected samples of meat (N=186) sold in the urban area of Ibagué (62 beef, 62 chicken, and 62 pork samples) and determined the T. gondii positivity percentage for each type of meat. Results: The study found an average of 18.8% positivity for all meat samples, pork having the highest percentage (22.5%; 14/62), followed by beef (19.3%; 12/62) and chicken (14.5%; 9/62). The best-amplified products were sequenced by macrogen and aligned with the B1 gene sequences in GenBank, thereby confirming their identity. Conclusions: This is the first study of T. gondii prevalence in meat for human consumption carried out in the city of Ibagué and the department of Tolima. All three types of meat sampled represent a risk for local human infection.

Toxoplasma gondii in South America: a differentiated pattern of spread, population structure and clinical manifestations.

Toxoplasma gondii is an obligate intracellular parasite belonging to the phylum Apicomplexa. It has a worldwide distribution and can infect a wide variety of intermediate hosts, including humans. In South America, toxoplasmosis shows high health impacts, and the incidence of the disease is frequently reported and more severe than in other regions, such as Europe. Although most T. gondii infections are asymptomatic, severe manifestations can occur in cases of congenital toxoplasmosis and immunocompromised individuals. In South America, the ocular disease in immunocompetent individuals is also frequently reported. Treatment for any clinical manifestation of toxoplasmosis consists of the combination of sulfadiazine (SDZ) and pyrimethamine (PYR). However, failures in the treatment of toxoplasmosis have been reported, especially in South America, suggesting the acquisition of resistance against SDZ and PYR. Another paradigm present in the literature is that once infected with T. gondii, the host is immunologically protected from further reinfections. However, some studies indicate cases of congenital transmission of T. gondii from immunocompetent pregnant women with chronic infection, suggesting the possibility of reinfection in humans. Thus, in this review, we will cover several aspects of South American T. gondii isolates, such as genetic characterization, disease manifestation, host reinfection and drug resistance.

Clinical and Multimodal Imaging Findings and Risk Factors for Ocular Involvement in a Presumed Waterborne Toxoplasmosis Outbreak, Brazil1.

In 2015, an outbreak of presumed waterborne toxoplasmosis occurred in Gouveia, Brazil. We conducted a 3-year prospective study on a cohort of 52 patients from this outbreak, collected clinical and multimodal imaging findings, and determined risk factors for ocular involvement. At baseline examination, 12 (23%) patients had retinochoroiditis; 4 patients had bilateral and 2 had macular lesions. Multimodal imaging revealed 2 distinct retinochoroiditis patterns: necrotizing focal retinochoroiditis and punctate retinochoroiditis. Older age, worse visual acuity, self-reported recent reduction of visual acuity, and presence of floaters were associated with retinochoroiditis. Among patients, persons >40 years of age had 5 times the risk for ocular involvement. Five patients had recurrences during follow-up, a rate of 22% per person-year. Recurrences were associated with binocular involvement. Two patients had late ocular involvement that occurred >34 months after initial diagnosis. Patients with acquired toxoplasmosis should have long-term ophthalmic follow-up, regardless of initial ocular involvement.

Association between IgG subclasses against Toxoplasma gondii and clinical signs in newborns with congenital toxoplasmosis.

BACKGROUND: The aim of this study was to evaluate the association between clinical signs of congenital toxoplasmosis and IgG subclasses found in newborns participating in the Minas Gerais State Neonatal Screening Program. METHODS: Neonates with confirmed congenital toxoplasmosis underwent standardized ophthalmologic evaluation, neuroimaging studies and hearing assessment, as well as enzyme-linked immunosorbent assay testing for total IgG and its subclasses (IgG1, IgG2, IgG3 and IgG4) against soluble (STAg) and recombinant (rSAG1 and rMIC3) antigens of Toxoplasma gondii. RESULTS: Newborns with congenital toxoplasmosis but without ocular lesions were more likely to present anti-rMIC3 total IgG when compared with those newborns with active or cicatricial retinochoroidal lesions. Detection of anti-rMIC3 IgG2 and IgG4 was associated with presence of retinochoroidal lesions and intracranial calcifications, with higher mean reactivity index values than unaffected newborns with congenital toxoplasmosis. Anti-STAg IgG3 was associated with newborns without neurologic damage. CONCLUSIONS: Specific subclasses of IgG antibodies reacting with recombinant antigens of T. gondii may serve as biomarkers of neurologic and ocular changes in newborns with congenital toxoplasmosis.

New naphthoquinones and an alkaloid with in vitro activity against Toxoplasma gondii RH and EGS strains.

The efficacy of three amino-terpenyl naphthoquinones and the alkaloid liriodenine were examined against tachyzoites and tissues cysts of the RH and EGS strains, respectively. Monolayers of 2C4 fibroblasts infected with tachyzoites of the RH strain were incubated with different concentrations of the compounds for 48 h. Specifically, 7-(4-methyl-3-pentenyl)-2-pyrrolidine-[1,4]-naphthoquinone (QUI-5), 6-(4-methyl-3-pentenyl)-2-pyrrolidine-[1,4]-naphthoquinone (QUI-6), 6-(4-methylpentyl)-2-pyrrolidine-[1,4]-naphthoquinone (QUI-11), and 8 h-benzo[g]-1,3-benzodioxolo[6,5,4-de]quinolin-8-one,9Cl-1,2-methylene dioxiaporfina (liriodenine) inhibited intracellular replication of T. gondii. The IC(50) values obtained for compounds QUI-5 and QUI-6 were 69.35 and 172.81 µM (i.e., 21.4 and 53.4 µg/mL), respectively. The naphthoquinone QUI-11 and liriodenine significantly inhibited intracellular replication of T. gondii. The IC(50) values obtained with these experiments were 0.32 and 0.07 µM (i.e., 0.1 and 0.02 µg/mL), respectively. Compounds QUI-5, QUI-6, QUI-11 and liriodenine demonstrated lower toxicity for 2C4 fibroblasts compared to atovaquone. In addition, cysts isolated from the brains of mice chronically infected with the EGS strain were exposed to the compounds. Infectivity of the cysts after incubation with the compounds was assessed by infection of mice. The data obtained showed that in vitro incubation with QUI-6, QUI-11 and liriodenine inhibited the infectivity of the bradyzoites. This activity was time- and concentration-dependent.

Identification of stage-specific proteins of Angiostrongylus vasorum (Baillet, 1866) Kamensky.

Sera of ten dogs infected with Angiostrongylus vasorum were analyzed by immunoblotting proteins of first stage larvae (L1) and adult parasites. The molecular weights (m.w.) of the principal L1 proteins identified by IgG, IgM, IgA, and IgE were 18-118 kDa and those of the adult parasite were 28-209 kDa. The L1 proteins had not been recognized by IgG, IgM, and IgA antibodies in sera of dogs naturally infected with Toxocara canis, Ancylostoma caninum, and Dipylidium caninum, although only weakly by IgE. Adult parasite proteins were recognized by antibodies in sera of dogs naturally infected with gastrointestinal helminths. Adult parasite proteins with m.w. of approximately 51, 63, 92, and 209 kDa recognized by IgG could be used for specific diagnosis of canine angiostrongylosis.