Sandra de Moraes Gimenes Bosco

Pythiosis is an underestimated and neglected disease in Brazil, both in horse breeders and in horses. The molecular detection of P. insidiosum in horses in the Brazilian Northeast represents a milestone in the epidemiology of equine pythiosis in the country. This study reports novel cases of equine pythiosis, diagnosed by molecular methods, in five states of Northeastern Brazil. Clinical samples were submitted to microbiological culture, DNA extraction, and nested-PCR for molecular detection of P. insidiosum. The nested-PCR successfully detected P. insidiosum in four out of five equine lesion samples, demonstrating higher sensitivity compared to microbiological culture, which confirmed the pathogen in only one case. These findings reinforce the effectiveness of molecular diagnosis for equine pythiosis compared to conventional methods. This is the first molecular confirmation of P. insidiosum in horses from Northeastern Brazil, emphasizing the need for broader surveillance and improved diagnostic approaches in neglected regions.

Cryptococcus is an opportunistic pathogen responsible for cryptococcosis, a fungal infection with high mortality, particularly in immunocompromised individuals. Accurate and rapid species identification is critical for effective diagnosis and treatment. This study developed and standardized a multiplex PCR protocol targeting the polymorphic PRP8 gene, a novel molecular marker proposed for differentiation of C. neoformans var. grubii, C. neoformans var. neoformans, and C. gattii. DNA was extracted from reference strains and primers were manually designed. The protocol demonstrated high specificity, with no amplification observed in negative controls, and reproducibility under optimized conditions, including an annealing temperature of 52 °C and 3% DMSO. This method is cost-effective, time-efficient, and robust, making it suitable for laboratories with limited resources. Compared to traditional targets such as coat genes and ribosomal RNA, the PRP8 gene offers enhanced resolution due to its intronic polymorphisms, allowing precise differentiation of molecular types. This innovation provides a valuable tool for clinical diagnostics and epidemiological studies, facilitating the understanding of geographic distribution, infection patterns and antifungal resistance profiles.

Antimicrobial drug resistance is a challenge to public health. Various microorganisms, including methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, carbapenemase-producing Klebsiella pneumoniae, Pythium insidiosum, and Candida auris, have developed resistance to commonly used antimicrobials in infection disease treatments. Consequently, there is an urgent need to explore and develop novel therapeutic drugs. Natural products, particularly medicinal plants, have received attention in the quest for innovative treatments for various diseases. Plantago major is a plant endowed with several biological properties, such as antibacterial, antifungal, and healing actions. This study aimed to investigate the antimicrobial activities of the methanolic extract obtained from the leaves of P. major. Microdilution assays were conducted to determine the minimum inhibitory concentration and minimum bactericidal and fungicidal concentration. Additionally, synergism with antimicrobial drugs was assessed using a time-kill curve analysis. A synergistic bactericidal interaction between the extract and imipenem was observed against carbapenemase-producing K. pneumoniae. For MRSA, a bacteriostatic synergism was noted in combinations of the extract with cephalotin and oxacillin. For C. auris, a fungistatic interaction was observed between the extract and amphotericin B. These results suggest the presence of bioactive compounds within the extract with therapeutic potential for combating infections caused by these microorganisms.

Pythium insidiosum is the etiological agent of pythiosis. This microorganism is a pathogenic oomycete that causes disease in several animal species, such as horses, dogs, cattle, sheep, goats, cats, birds and also humans. It is a disease characterized by lesions in the cutaneous/subcutaneous tissues in the form of exudative granulomas, the formation of “kunkers” being common in horses. It can also cause damage in gastrointestinal tract, vascular, ocular, and disseminated to internal organs. The diagnostic methods are, in addition to clinical signs and patient history, culture and identification of the agent, serological tests (immunodiffusion, ELISA, Western blot, hemagglutination and immunochromatographic tests), histopathological, immunohistochemical, molecular tests and recently, proteomic analysis, like MALDI-TOF. The treatment is based on surgical exeresis of the lesion with margins free of the pathogen, and this treatment is only possible in regions without involviment of important anatomical structures; use of drugs such as Amphotericin B and Potassium Iodide and immunotherapy as a way to modulate the host immune response. There is no predisposing factor for acquiring the disease, the main risk being staying in stagnant water with the presence of plant material. Thus, the main form of infection is to avoid, when possible, keeping animals inside ponds, rivers/streams or dams for long periods, as well as using personal protective equipment on humans when they need to enter such environments. Pythium insidiosum es el agente etiológico de la pitiosis. Este microorganismo es un oomiceto patógeno que causa enfermedad en varias especies animales, como caballos, perros, bovinos, ovinos, caprinos, felinos, aves y también humanos. Es una enfermedad caracterizada por lesiones en el tejido cutáneo/subcutáneo en forma de granulomas exudativos, siendo frecuente la formación de kunkers en los caballos. También puede causar lesiones gastrointestinales, vasculares y oculares y diseminarse a los órganos internos. Los métodos diagnósticos son, además de los signos clínicos y la historia del paciente, el cultivo e identificación del agente, las pruebas serológicas (inmunodifusión, ELISA, Western blot, hemaglutinación y prueba inmunocromatográfica), la histopatología, la inmunohistoquímica, las pruebas moleculares y, recientemente, el análisis proteómico, como el MALDI-TOF. El tratamiento se basa en la escisión quirúrgica de la lesión con márgenes libres de patógenos, y este tratamiento solo es posible en regiones sin compromiso de estructuras anatómicas importantes; uso de fármacos como la anfotericina B y el yoduro de potasio y la inmunoterapia como forma de modular la respuesta inmunitaria del huésped. No existe un factor predisponente para adquirir la enfermedad, siendo el principal riesgo permanecer en aguas estancadas con presencia de material vegetal. Así, la principal forma de infección es evitar, en lo posible, mantener a los animales dentro de estanques, ríos/arroyos o represas por períodos prolongados, así como usar equipo de protección personal en humanos cuando necesitan ingresar a dichos ambientes. Pythium insidiosum é o agente etiológico da pitiose, sendo ele um microrganismo da classe dos oomicetos, patogênico e causador da pitiose em várias espécies animais, incluindo humanos. A doença é caracterizada por lesões no tecido cutâneo/subcutâneo, lesões gastrointestinais, vasculares, oculares, podendo se disseminar para órgãos internos. A forma cutânea gera granulomas exsudativos, sendo comum a formação de “kunkers”, um material necrótico de coloração amarelada, apenas nos equídeos. O diagnóstico é feito através dos sinais clínicos e histórico do paciente, cultura e identificação do agente e alguns testes como os sorológicos (imunodifusão, ELISA, Western blot, hemaglutinação e teste imunocromatográfico), histopatológico, imunohistoquímica, testes moleculares e recentemente, análises proteômicas, como o MALDI –TOF. O tratamento baseia-se na exérese cirúrgica da lesão com margens livre do patógeno, sendo apenas possível em regiões sem comprometimento de importantes estruturas anatômicas; uso de medicamentos como Anfotericina B e Iodeto de Potássio e a imunoterapia como forma de modular a resposta imune do hospedeiro. Não existe fator predisponente para a aquisição da doença, sendo o principal risco a permanência em águas estagnadas com presença de material vegetal. Dessa forma, a principal forma de infecção é evitar, quando possível, a manutenção por longos períodos de animais no interior de lagoas, rios/riachos ou açudes, bem como utilizar equipamentos de proteção individual em humanos quando necessitarem adentrar em tais ambientes.

The high complexity of the oral microbiota of healthy dogs and the close exposure of humans to companion animals represent a risk of the transmission of potential zoonotic microorganisms to humans, especially through dog bites, including multidrug-resistant ones. Nonetheless, a limited number of comprehensive studies have focused on the diversity of the microorganisms that inhabit the oral cavities of healthy dogs, particularly based on modern molecular techniques. We investigated bacterial and fungal organisms in the oral cavities of 100 healthy dogs based on a combination of conventional and selective microbiological culture, mass spectrometry (MALDI-TOF MS), and next-generation sequencing. In addition, in vitro antimicrobial susceptibility patterns of isolates and mecA resistance gene were assessed. A total of 213 bacteria and 20 fungi were isolated. Staphylococcus pseudintermedius (40/100 = 40%), α-hemolytic Streptococcus (37/100 = 37%), and Pasteurella stomatis (22/100 = 22%) were the most prevalent bacteria diagnosed by microbiological culture and MALDI-TOF MS, whereas Aspergillus (10/100 = 10%) was the most common fungi identified. Based on next-generation sequencing of selected 20 sampled dogs, Porphyromonas (32.5%), Moraxella (16.3%), Fusobacterium (12.8%), Conchiformibius (9.5%), Bergeyella (5%), Campylobacter (3.8%), and Capnocytophaga (3.4%) genera were prevalent. A high multidrug resistance rate was observed in Staphylococcus pseudintermedius isolates, particularly to azithromycin (19/19 = 100%), penicillin (15/19 = 78.9%), and sulfamethoxazole/trimethoprim (15/19 = 78.9%). In addition, the mecA resistance gene was detected in 6.1% (3/49) of coagulase-positive staphylococci. Here, we highlight the microbial complexity of the oral mucosa of healthy dogs, including potential zoonotic microorganisms and multidrug-resistant bacteria, contributing with the investigation of the microbiota and antimicrobial resistance patterns of the microorganisms that inhabit the oral cavity of healthy dogs.

The study aimed to describe the clinicopathological aspects of 37 cases of pythiosis, 34 in horses and three in mules, from properties located in the Amazon biome of Pará, Brazil. The clinical signs observed in the animals were weakness, poor-to-regular nutritional status, pale mucous membranes, itching at the lesion site, and lameness when the limbs were affected. The lesions were located on the lips, nostrils, rib region, thoracic and abdominal walls, scapular, distal limbs, foreskin, perineum and udder. Macroscopically, ulcerative and granulation-tissue-like masses were observed, with fistulous tracts filled with yellowish and foul-smelling serosanguinous discharges. In the biopsy, it was possible to visualize white and firm areas with foci of yellowish necrotic material and hardened masses, called kunkers. The histopathological examination showed a pyogranulomatous inflammatory reaction with the presence of Pythium insidiosum hyphae, which were impregnated with black, confirming the diagnosis of pythiosis in equids in the Amazon biome, being the first report of the disease in mules in the region.

Pythiosis, whose etiological agent is the oomycete Pythium insidiosum, is a life-threatening disease that occurs mainly in tropical and subtropical countries, affecting several animal species. It is frequently found in horses in Brazil and humans in Thailand. The disease is difficult to diagnose because the pathogen’s hyphae are often misdiagnosed as mucoromycete fungi in histological sections. Additionally, there is no specific antigen to use for rapid diagnosis, the availability of which could improve the prognosis in different animal species. In this scenario, we investigated which P. insidiosum antigens are recognized by circulating antibodies in horses and humans with pythiosis from Brazil and Thailand, respectively, using 2D immunoblotting followed by mass spectrometry for the identification of antigens. We identified 23 protein spots, 14 recognized by pooled serum from horses and humans. Seven antigens were commonly recognized by both species, such as the heat-shock cognate 70 KDa protein, the heat-shock 70 KDa protein, glucan 1,3-beta-glucosidase, fructose-bisphosphate aldolase, serine/threonine-protein phosphatase, aconitate hydratase, and 14-3-3 protein epsilon. These results demonstrate that there are common antigens recognized by the immune responses of horses and humans, and these antigens may be studied as biomarkers for improving diagnosis and treatment.

Pythiosis is a disease caused by the oomycete Pythium insidiosum, mainly reported in equines, dogs, and humans and directly transmitted through contaminant zoospores in aquatic environments. We report the first outbreak of equine pythiosis in five equines. Wound samples were submitted for diagnostic testing including mycological culture and nested PCR. Treatment approaches consisted of conventional and alternative therapies. Microbiological analyses were performed using water samples from the riverbanks close to where the animals had grazed. All animals were positive for P. insidiosum cultures, and two animals responded successfully to alternative therapy (ozone‐therapy). After culture and molecular analysis of environmental samples, the presence of P. insidiosum in one section of the Tietê River was confirmed through a 99% sequence identity. Phylogenetic analyses using the cytochrome‐oxidase II gene showed that the animal isolates clustered in clade I and the environmental isolates clustered in clade III. Although the environmental and wound isolates belonged to different genetic clades, we concluded that the Tietê River is an important source of infection by P. insidiosum and that research concerning environmental isolation of P. insidiosum from rivers and lakes should be strongly facilitated in Brazil.

Armadillos are specialist diggers and their burrows are used to find food, seek shelter and protect their pups. These burrows can also be shared with dozens of vertebrate and invertebrate species and; consequently, their parasites including the zoonotics. The aim of this study was to diagnose the presence of zoonotic parasites in four wild‐caught armadillo species from two different Brazilian ecosystems, the Cerrado (Brazilian savanna) and the Pantanal (wetland). The investigated parasites and their correspondent diseases were: Toxoplasma gondii (toxoplasmosis), Trypanosoma cruzi (Chagas disease), Leishmania spp., (leishmaniasis), Paracoccidioides brasiliensis (Paracoccidioidomicosis) and Mycobacterium leprae (Hansen’s disease). Forty‐three free‐living armadillos from Pantanal and seven road‐killed armadillos from the Cerrado were sampled. Trypanosoma cruzi DTU TcIII were isolated from 2 out of 43 (4.65%) armadillos, including one of them also infected with Trypanosoma rangeli. Antibodies anti‐T. gondii were detected in 13 out of 43 (30.2%) armadillos. All seven armadillos from Cerrado tested positive for P. brasiliensis DNA, in the lungs, spleen, liver fragments. Also, by molecular analysis, all 43 individuals were negative for M. leprae and Leishmania spp. Armadillos were infected by T. cruzi, T. rangeli, P. brasiliensis, and presented seric antibodies to T. gondii, highlighting the importance of those armadillos could have in the epidemiology of zoonotic parasites.

Bats are essential to the global ecosystem, but their ability to harbour a range of pathogens has been widely discussed, as well as their role in the emergence and re‐emergence of infectious diseases. This paper describes the first report of coinfection by two zoonotic agents, rabies virus (RABV) and the fungus Histoplasma suramericanum in a bat. The bat was from the Molossus molossus species, and it was found during the daytime in the hallway of a public psychiatric hospital in a municipality in São Paulo State, southeastern Brazil. RABV infection was diagnosed by the direct fluorescent antibody test and mouse inoculation test. The fungus was isolated by in vitro culture. Both diagnoses were confirmed by molecular techniques. Phylogenetic analysis showed that the fungus isolate had proximity to H. suramericanum in the Lam B clade, while the RABV isolate was characterized in the Lasiurus cinereus lineage. Since the M. molossus bat was found in a peri‐urban transition area (urban/peri‐urban), the possibility of cross‐species transmission of this RABV lineage becomes more plausible, considering that this scenario may provide shelter for both M. molossus and L. cinereus. These are relevant findings since there has been an increase in bat populations in urban and peri‐urban areas, particularly due to environmental modifications and anthropogenic impacts on their habitat. Thus, the detection of two zoonotic agents in a bat found in a public hospital should raise awareness regarding the importance of systematic surveillance actions directed towards bats in urban areas.