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This study aimed to research Toxoplasma gondii DNA in 102 samples of raw bovine milk from expansion tanks, in small properties located in different cities of the Midwest region of São Paulo, Brazil. For this, polymerase chain reaction (PCR) was performed with the primers TOX4/TOX5 for cPCR (conventional PCR), TgNP1/TgNP2 gene for nested PCR and the Tg18s58F/Tg18s348R for nested PCR. It was possible to detect T. gondii DNA in 18 (17.65 %) milk samples from the 102 tanks, corresponding to 4.90 % for TOX4/TOX5 primers, 12.74 % for TgNP1/TgNP2 gene and 0.98 % for Tg18s58F/Tg18s348R gene. The results showed that the TgNP1 and TgNP2 genes were more efficient to detect T. gondii DNA, and also indicated the importance of raw bovine milk as a source of human infections caused by this protozoan, being a public health problem. It is important to continue studies involving T. gondii from bovine milk considering the need for proper pasteurization, and for better comprehension regarding the epidemiology of this protozoan., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background and Objectives: Feline leishmaniasis (FeL) is caused by several species of parasites of the genus Leishmania . The disease can occur with the presence or absence of clinical signs, similar to those observed in other common infectious diseases. In endemic regions for FeL, the infection has been associated with dermatological lesions. Therefore, considering the search for less invasive and more effective diagnostic techniques, we aimed to investigate the presence of Leishmania spp. in domestic cats through Polymerase Chain Reaction (PCR) and high-resolution melting (HRM) analyses of conjunctival, oral, and nasal epithelial cells, and we detected the presence of anti- Leishmania IgG antibodies from serological techniques of the Immunofluorescent Antibody Test (IFAT) and ELISA., Methods: The PCR and HRM for detection of Leishmania spp. were performed on 36 samples of epithelial cells from the conjunctiva of male and female cats, collected using sterile swabs. The serological tests IFAT and ELISA were also performed., Results: The prevalence of Leishmania donovani infection was 11.1% (4/36) by PCR assay, and those results were confirmed for Leishmania species using the HRM technique. Twenty-four cats (24/36 = 66.7%) were reactive to the IFAT and twenty-two cats were reactive by the ELISA technique (22/36 = 61.1%)., Interpretation and Conclusions: The use of conjunctival swabs was shown to be a non-invasive, practical, and easy-to-perform technique, and in addition to the genetic sequencing and HRM, it was able to identify the parasitic DNA of L. donovani in cats. This technique can be used for screening diagnosis in future epidemiological surveys of FeL and can be used as a complement to clinical and/or serological tests, as well as associating the clinical history of the animal, for the diagnostic conclusion.

The performance of a commercial immunofluorescence assay (IFA commercial), an in-house immunofluorescence assay (IFA in-house ) and an indirect enzyme-linked immunosorbent assay (ELISA) were evaluated in the detection of antibodies anti- C. burnetii in the serum of Q fever patients and persons without the disease. For the study, seropositive and seronegative samples for Q fever ( n = 200) from a serum bank of the Instituto Adolfo Lutz in Brazil were used. Commercial IFA was considered in this study as the gold standard for diagnosing Q fever. The in-house IFA demonstrated good agreement with the commercial test, showing high sensitivity (91%) and specificity (97%) compared to the gold standard, with a Kappa coefficient of 0.8954. The indirect ELISA test showed lower agreement with the gold standard, showing low sensitivity (67%), although the specificity of the technique was high (97%) and the Kappa coefficient was moderate (0.6631). In-house IFA is an excellent alternative for diagnosing Q fever.

Trichosporonosis corresponds to a systemic fungal disease that leads to high mortality rates and is frequently associated with medical devices. It affects immunosuppressed patients in particular and is strongly linked to acquired human immunodeficiency, organ and tissue transplants, and malignant hematologic diseases such as leukemia and lymphomas. Trichosporon infections have been increasingly reported worldwide; however, little information is available either about their characteristics or the causative microorganism. Thus, the aims of the present study were: to investigate 59 yeasts of the genus Trichosporon by verifying the biofilm formation capacity of isolates; to analyze the susceptibility patterns of planktonic cells against the antifungals fluconazole, itraconazole, amphotericin-B, voriconazole, and caspofungin by comparing European Committee for Antimicrobial Susceptibility Testing (EUCAST) broth microdilution technique with the commercial method Etest; and to assess the susceptibility patterns of biofilm cells (sessile) against the same antifungals through broth microdilution. The ability to form biofilm on the surface of polystyrene plates was noted for all isolates, and 54.3% of samples were considered strong producers. Comparison between the antifungal susceptibility techniques evidenced that Etest showed higher and discordant minimum inhibitory concentrations (MICs) from those obtained by the microdilution method, especially for fluconazole, itraconazole, and caspofungin. Considering the susceptibility of biofilms, most species had high MIC50 and MIC90 against the tested antifungals, showing 4-to-66-fold higher concentrations for amphotericin B and 2-to-33-fold greater concentrations for caspofungin. These results highlight the importance of further studies with Trichosporon spp. for comparison between laboratory findings and in vivo response, considering both the susceptibility tests and the behavior of biofilm cells against drugs., (© The Author(s) 2023. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Q fever and brucellosis are zoonoses that cause fever and other systemic clinical signs in humans; their occurrences are neglected and the differential diagnosis for some diseases is disregarded. This study aimed to investigate the seropositivity for Coxiella burnetii and Brucella spp. antibodies in patients suspected of dengue from 38 municipalities in the state of São Paulo, Brazil. The samples (n = 604) were obtained by convenience from the Adolfo Lutz Institute serum bank. Sera were subjected to an indirect immunofluorescence assay (IFA) using in-house and commercial diagnostic protocols to evaluate C. burnetii positivity. For Brucella spp., sera were subjected to rapid plate serum agglutination with buffered acidified antigen (AAT), slow tube serum agglutination (SAL), and 2-mercaptoethanol (2-ME) techniques. Associations and statistical inferences of the results were performed by logistic regression according to the clinical and demographic variables collected from the patients. Statistical analyses were performed using Statistical Analysis Software (SAS) and associations were considered when p value was <0.05. In all, 129 patients showed positive results for Q fever, indicating a seropositivity of 21.4% (95% CI 18.15-24.85). Patients with 14-20 days of symptoms had 2.12 (95% CI 1.34-3.35) times more chances of being seropositive for Q fever than patients with 7-13 days, and patients with 21-27 days of fever had 2.62 (95% CI 1.27-5.41) times more chances of being seropositive for Q fever than patients with 7-13 days. For the other variables analyzed, there were no significant associations between the groups. No positivity for brucellosis was observed. This is the most comprehensive study of people seropositive for Q fever in São Paulo state and provides additional data for the medical community in Brazil. It is suggested that Q fever may be an important differential diagnosis of febrile illnesses in the region, demanding the government's attention and investment in health., Competing Interests: The authors have declared that no competing interests exist.

Wild nonhuman primates (NHP) are considered natural hosts of a protozoan parasite from the genus Leishmania, the etiological agent of leishmaniasis. It is important to study the population of this infectious agent in zoo animals to establish surveillance and control mechanisms in Sorocaba through the application of a One Health approach, this is where human-animal-environment health and disease interface and can aid in the protection of endangered species. This study aimed to identify Leishmania infantum and Leishmania braziliensis in NHP living in a city where leishmaniasis is endemic. DNA was extracted from 48 NHP and analyzed using polymerase chain reaction primers that are specific for the species L. infantum and L. braziliensis. The results of our research revealed the first report of L. infantum and L. braziliensis naturally infecting primates at Sorocaba zoo. One primate from the species Plecturocebus vieirai was positive for L. infantum and five primates (four Alouatta caraya and one Ateles chamek) were positive for L. braziliensis. This indicates a possible role of these animals on the maintenance of these parasites., (© 2022 Wiley Periodicals LLC.)

Aims: To evaluate the physicochemical and microbiological quality of dialysis water and dialysate samples from haemodialysis centres., Methods and Results: Samples were fortnightly collected from three haemodialysis centres in Bauru City, Brazil, between July 2017 and June 2018, at the stages of post-reverse osmosis, reuse and dialysate. Analyses included determination of conductivity, fluoride, nitrate and sulphate; test for total coliform bacteria; count of heterotrophic bacteria; count and identification of non-fermenting gram-negative bacilli (NFGNB); drug susceptibility test; biofilm formation capacity; and genetic similarity among some isolated NFGNB. Of the analysed samples, only 4/72 (5.6%) had conductivity values ≥10 mS/cm, 4/216 (1.9%) presented total coliforms and 1/216 (0.5%) had heterotrophic bacteria count >100 CFU/ml. NFGNB were isolated from 99/216 (45.8%) samples, and the major identified micro-organisms included Herbaspirillum aquaticum/huttiense, Brevundimonas aurantiaca, Cupriavidus metallidurans, Pseudomonas aeruginosa and Ralstonia insidiosa. Isolates of P. aeruginosa and Burkholderia cepacia complex were sensitive to most antimicrobials and, together with isolates of Ralstonia insidiosa and Ralstonia pickettii, showed strong biofilm formation capacity. Some isolates expressed the same electrophoretic profile on pulsed-field gel electrophoresis, indicating the persistence of bacterial clones in the systems over time., Conclusions: NFGNB were observed in several dialysis water and dialysate samples from all investigated centres, which may represent a risk to the health of patients., Significance and Impact of the Study: Regular inclusion of actions for NFGNB control and monitoring in haemodialysis fluids are suggested for greater safety of the dialytic process., (© 2022 Society for Applied Microbiology.)

Trichosporon spp. are widely distributed in the nature, comprising species that inhabit different ecological niches and can be found in the water, soil, and body surface of animals and humans. Such microorganisms have been classically associated with superficial infections; however, in the last decades, they have also been related to disseminated infections in immunocompromised patients, behaving as opportunistic agents, which demands rapid and accurate species identification for efficient therapy. Concordance level between the traditional phenotypic method and the molecular technique (gold standard) in the identification of all 59 Trichosporon samples was 59.3%. Identification concordance between MALDI-TOF spectrometry and the molecular technique was 71.2%. No isolate of environmental origin was identifiable by MALDI-TOF mass spectrometry (MS), and 100% of such environmental isolates were discordant for IGS region sequencing and phenotypic characterization. Both comparisons evidenced greatest concordance in the identification of T. asahii. The species T. debeurmannianum, T. dermatis, T. venhuisii and T. insectorum were not properly identified by both MALDI-TOF MS and the phenotypic technique. MALDI-TOF MS, in particular, seems to be appropriate to investigate yeasts of the genus Trichosporon; however, database updates are still necessary, especially for species that are not common in the clinical routine. With the aim of helping understand the aspects involved in early and accurate diagnosis of infections caused by this opportunistic agent, the present study compared the phenotypic, molecular (IGS region) and mass-spectrometry (MALDI-TOF) identification of 59 yeasts of the genus Trichosporon which had clinical and environmental origin and were kept in a mycology collection., (© The Author(s) 2021. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Toxoplasmosis occurs worldwide causing economic losses to the animal production and problems to the public health. The study aimed to detect Toxoplasma gondii and Sarcocystis spp.in 141 meat products from commercial meat cuts of pork, beef, and kibbeh sold in commercial markets from Botucatu, SP, Brazil. Samples were bioassayed in mice to isolate the parasite, and the parasite DNA detected by PCR targeting the 529 base pairs repeat element region (PCR-529-bp). All samples resulted negative on bioassay, whereas PCR positive for 9 (6,38%), distributed as 5/48 beef, 3/49 pork, and 1/44 kibbeh. PCR-positive were investigated for the the parasite genotype using multiplex-, nested-, and RFLP-PCR for 11 markers (SAG1, 5'-3'SAG2, alt.SAG2, SAG3, B-TUB, GRA6, L358, c22-8, c29-6, PK1, Apico). Complete genotype was determined on just one PCR-positive sample that matched MAS, TgCkBr89 and TgCkBr147 isolates already identified. In addition, nested- and RFLP-PCR targeting 18S rRNA was run for all PCR-positive samples and, the products, sequenced and aligned to the GenBank at NCBI website. Four samples showed 100% homology with T. gondii (GenBank #L37415.1), three with Sarcocystis hominis (GenBank #AF006471.1), two Sarcocystis cruzi (GenBank #AF176934.1), and one Sarcocystis hirsuta (GenBank #AF006469.1), indicating the circulation of T. gondii and Sarcocystis spp.

A paleoparasitological analysis was carried out on a large coprolite assigned to a carnivoran mammal, recovered from the Municipality of Uruguaiana, in the western region of the State of Rio Grande do Sul, Brazil, where the Upper Pleistocene Touro Passo Formation crops out. For this, an individual sample was extracted from the specimen using an electric drill, dissociated with 10% hydrochloric acid solution, washed with distilled water, and sifted through a 500 mesh Tyler sieve. After laboratory processing, the sediment retained on the sieve was mixed with glycerin and examined by optical microscopy, which revealed the presence of 14 protozoan oocysts and three nematode eggs. The morphological characteristics of the oocysts (i.e., spherical shape, thick-walled, internal zygote apparently at the beginning of sporulation, as well as their size) and of the eggs (i.e., ovoidal shape, rounded ends, smooth surface, thin-shelled, embryo in their interior, along with their morphometry) suggest that these specimens belong respectively to the orders Eucoccidiorida and Strongylida (Family Ancylostomatidae) represented by several parasitic species of the alimentary tract of modern carnivore. This is the first record of paleoparasites discovered in a vertebrate host from the Touro Passo Formation.

Background: The early symptoms of leptospirosis and dengue fever are difficult to distinguish and can cause diagnostic confusion. Due to the large dengue epidemics that has occurred in Brazil in recent years, it is possible that cases of leptospirosis were unreported. Therefore, we performed a retrospective study to detect leptospirosis in patients who were tested for dengue, but whose laboratory diagnoses were negative., Methods: Sera samples from 2,017 patients from 48 cities located in the central region of São Paulo state, Brazil, were studied. All samples were subjected to the microscopic agglutination test (MAT), 305 of which were taken from patients five days or less since the onset of symptoms, and were additionally subjected to real-time polymerase chain reaction (PCR)., Results: The overall prevalence of leptospirosis cases was 21 (1.04%), with 20 through MAT (18 for Icterohaemorrhagiae and two for the Cynopteri serogroup) and one through PCR (amplicon sequencing compatible with Leptospira interrogans ). According to previously established criteria, eight cases of leptospirosis were classified as "confirmed" and 13 as "probable". The Brazilian notification system for health surveillance had no records for 16 patients positive for leptospirosis and, thus, they were considered unreported cases. Statistical analyses revealed that the prevalence of leptospirosis was higher in men (1.56%) than in women (0.56%), and the mean age was higher in positive patients (43.7 years) than in negative ones (32.3 years)., Conclusion: The results indicated that patients suspected of dengue fever had evidence of leptospirosis or Leptospira infection, and most of these cases were unreported in the Brazilian notification system. The high burden of dengue may contribute to the misdiagnosis of leptospirosis, and health professionals should increase their awareness of leptospirosis as an important differential diagnosis of patients with suspicion of dengue., Competing Interests: Competing interests: The authors declare no conflicts of interest.

We evaluated 345 wild animals from southern and south-eastern Brazil to understand their role in vaccinia virus (VACV) transmission cycle. VACV DNA was detected in rodents, marsupials, chiroptera and cingulate, expanding the knowledge of VACV host range in wildlife that could potentially act as source of infection in rural and urban areas., (© 2020 Blackwell Verlag GmbH.)

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., (© 2019 Blackwell Verlag GmbH.)

This is the first report of the yeast Apiotrichum veenhuisii (formerly Trichosporon veenhuisii ) causing disease in humans; its virulence and in vitro behavior against antifungals were also studied. The sample was isolated from biopsy fragments of disseminated lesions on the skin of a pediatric patient with acute myeloid leukemia. The studied virulence factors evidenced that the strain tested negative for secretion of the enzymes proteinase, phospholipase, and hemolysin. The isolate was characterized as low biofilm producer. Except for amphotericin B and voriconazole, the sample presented high minimum inhibitory concentration values against azole and echinocandins.

Leishmaniasis is considered a parasitic disease that still causes serious consequences for mankind, because it presents a high mortality rate worldwide. Considered multi-hosts, the parasites of the genus Leishmania are able of infecting a wide variety of animal species. The dog was considered the main source of infection of visceral leishmaniasis (VL), in the urban area. However, the role of other animal species in the epidemiological cycle of the disease, such as cattle, remains unclear. Therefore, the aim of the present study was to evaluate the occurrence of Leishmania spp. in 100 bovines (Bos taurus) from an area endemic for canine VL, using blood culture and molecular analysis. By the sequencing analysis, one sample showed 100% similarity with Leishmania infantum. The results provide the first case of L. infantum isolation in one bovine from the periurban areas of Bauru, state of São Paulo, Brazil.

Leishmaniasis is a vector-borne parasitic protozoan infection that affects mammals and involves a complex epidemiology. Although dogs are considered the main reservoir in zoonotic visceral leishmaniasis (VL), the possible presence of other mammalian species acting as reservoirs has been associated as a possible cause of lack of success in the control of human VL in many endemic areas. The knowledge about natural infections of some species is still scarce, such as nonhuman primates (NHP), especially from the genus Callithrix (marmosets). We investigated the infection by Leishmania (Leishmania) infantum, the agent of VL in the Americas, in 26 marmosets captured monthly, from April 2014 to March 2015, in an environmentally protected area (EPA) in Southeastern Brazil. The EPA has undergone significant environmental changes and has a transmission focus of canine VL since 2009. Serology was performed through the direct agglutination test, which detected low antibody titers in seven marmosets (7/26; 26.9%, 95% confidence interval 9.9-44.0), being five Callithrix penicillata (black-tufted-ear marmoset) and two Callithrix jacchus (white-tufted-ear marmoset). The presence of the DNA of Leishmania was investigated in blood and skin samples by PCR and genetic sequencing. This is the first report of the detection of L. (L.) infantum in the skin of a marmoset, which was verified in a sample from one C. penicillata. The results demonstrate the natural infection of marmosets by L. (L.) infantum and may suggest the participation of these animals as hosts in the parasite's transmission cycle in the EPA. However, more comprehensive studies are needed to elucidate their role on the VL epidemiology in this area and also in different endemic areas, especially because these NHP are increasingly in contact with humans and domestic animals, particularly due to environmental changes.

Epidemiological studies on endemic mycosis can be improved using molecular biology techniques to elucidate the role of bats as reservoirs and vectors of pathogenic fungi for infection of other animals and humans. The objective of this study was to explore the presence of Histoplasma capsulatum, Cryptococcus spp. and Paracoccidioides brasiliensis in insectivorous, frugivorous and nectarivorous bats collected in urban areas. We analysed 172 bats collected by the Epidemiological Surveillance Agency in 12 municipalities of the Midwest region of the state of São Paulo, Brazil. Spleen, liver, intestine and lung samples were subjected to microbiological culture and nested PCR analyses. Prevalence of H. capsulatum infection was 8.1% (14/172), with one bat found to be positive by fungal culturing, 12 positive by nested PCR and one positive by both methods. Two insectivorous bats were found positive by nested PCR for Cryptococcus spp., one in the spleen and the other in the spleen and lung. Two insectivorous bats showed natural infection by P. brasiliensis, in the spleen of one bat and the spleen and liver of the other. Our results reinforce the importance of bats as fungal dispersers in urban environments and the importance of constant epidemiologic surveillance because these synanthropic animals are in close contact with humans and animals., (© 2018 Blackwell Verlag GmbH.)

Bats are considered to play a significant role in the epidemiology of histoplasmosis, worldwide. We investigated the occurrence of H. capsulatum in lung samples from 89 bats, from urban areas in Southeastern Brazil, using nested PCR based on ribosomal DNA. Fungal DNA was detected in 31/89 samples (34.8%), of which 13/31 were Molossids (41.9%), 4/31 Eumops spp. (12.9%), 2/31 Artibeus lituratus (6.5%), and 12/31 others (38.7%). This is the first report of natural infection by H. capsulatum in A. lituratus in Southeastern Brazil, which reinforces the importance of these synanthropic animals in the epidemiology of histoplasmosis in urban areas.

Some animals have an important relationship with fungal infections, and searching for pathogens in animal samples may be an opportunity for eco-epidemiological research. Since studies involving wildlife are generally restricted, using samples from road kills is an alternative. The aim of this study was to verify whether pathogenic fungi of public health importance occur in wildlife road kills from Santa Catarina State, Brazil. Organ samples (n = 1063) from 297 animals were analysed according to Polymerase Chain Reaction (PCR) using universal primers to detect fungi in general and, subsequently, using primers specific to Paracoccidioides brasiliensis, Histoplasma capsulatum and Cryptococcus spp. There were 102 samples positive for fungal species. Eight samples were positive for P. brasiliensis, three samples were positive for Cryptococcus spp. and one sample had coinfection by these two fungi. No sample was positive for Histoplasma spp. according to the molecular detection. Genetic sequencing allowed the identification of Fungal sp. in 89 samples, Cryptococcus neoformans in two samples and Aspergillus penicillioides in three samples. This study shows the importance of wild animals in the epidemiology of fungal infections and assists in the mapping of pathogen occurrence in a region that was not previously evaluated., (© 2018 Blackwell Verlag GmbH.)

Leishmaniasis is a disease with ample clinical spectrum and epidemiological diversity and is considered a major public health problem. This article presents an overview of the transmission cycles, host-parasite interactions, clinical, histological and immunological aspects, diagnosis and treatment of various forms of the human disease.

Background: Canine visceral leishmaniasis (CVL) is a worldwide parasitic zoonosis caused by Leishmania (Leishmania) infantum around the world. Canids are the definitive hosts and sand flies the intermediate hosts., Objective: To test the hypothesis that a new species-specific primers (Lch14:Lch15, targeting a multiple alignment for L. infantum kDNA minicircle) is an efficient diagnostic tool for L. infantum., Methods: The presence of L. infantum DNA was assessed in blood samples of 69 stray dogs using the conventional PCR (cPCR) and quantitative PCR (qPCR). Additional 50 lymph nodes and 50 bone marrow samples (positive and negative samples for parasitological tests) from dogs from endemic and nonendemic areas for CVL were also used., Results: L. infantum strains, and all positive lymph node and bone marrow samples for parasitological test gave positive results for cPCR and qPCR, presenting analytical sensitivity of ∼10 0 parasite mL -1 . For the blood samples, 40/69 (58%; CI 95%; 46%-69%) resulted positive for L. infantum in both tests. All positive samples were confirmed by sequencing., Conclusion: This study showed the importance of the specific detection of L. infantum based on species-specific primers by molecular techniques, highlighting the application as a confirmation method in epidemiological studies and to adopt the best control measures.

Leishmaniasis affect millions of people, causing morbidity and mortality, especially in developing tropical and subtropical countries. Unfortunately, the possibilities of treatment for these infections are still quite limited and most of the available drugs present serious side effects. The objective of this paper was to evaluate the therapeutic role of amiodarone and itraconazole in the treatment of cutaneous leishmaniasis caused by Leishmania (Leishmania) amazonensis. In order to perform this evaluation, hamsters were infected with 1 × 10 6 metaciclic promastigotes of the parasite in the hind footpad and, after the onset of the lesions, were treated with glucantime, amiodarone, itraconazole, glucantime and amiodarone, glucantime and itraconazole or amiodarone and itraconazole. The treatments' efficacy was evaluated per analysis of the size of the cutaneous lesions and by parasitic investigation of the infected foot (by histopathological examination and PCR) and possible side effects were analyzed taking into account the weight of the animals and some biochemical and metabolic parameters (glucose, urea, creatinine, AST, ALT and ALP). The results have shown that, in hamsters, amiodarone and itraconazole, either used isolated or in combination, are unable to stop the development of cutaneous lesions caused by L. (L.) amazonensis, but improve the activity of glucantime in the treatment of these lesions and seem to present no evident side effects. More studies are necessary in order to investigate the clinical potential of these combinations, so there can be the possibility of broadening the therapeutic options available, especially in resistant cases., (Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

Background: Leishmaniasis is a rapidly expanding zoonosis that shows increasing urbanization. Concern exists regarding the role of wildlife in visceral leishmaniasis (VL) transmission, due to frequent natural or anthropogenic environmental changes that facilitate contact between wildlife, humans and their pets. The municipality of Campinas, in southeastern Brazil, initially recorded VL in 2009, when the first autochthonous case was confirmed in a dog living in an upscale residential condominium, located inside an environmentally protected area (EPA). Since then, disease transmission remains restricted to dogs inhabiting two geographically contiguous condominiums within the EPA., Methodology/principal Findings: We conducted a cross-sectional study of the VL focus to investigate Leishmania spp. infection in domestic dogs, wild mammals and sand flies using molecular tools and recommended serological techniques. Canine seroprevalences of 1.5% and 1.2% were observed in 2013 and 2015, respectively. Six insect species, confirmed or suspected vectors or potential transmitters of Leishmania, were identified. Two specimens of the main L. (L.) infantum vector in Brazil, Lutzomyia longipalpis, were captured in the EPA. Natural infection by L. (L.) infantum was recorded in one Expapillata firmatoi specimen and two Pintomyia monticola. Natural infection by L. (L.) infantum and Leishmania subgenus Viannia was also detected in two white-eared opossums (Didelphis albiventris), a known reservoir of VL. Geographical coordinates of each sampling of infected animals were plotted on a map of the EPA, demonstrating proximity between these animals, human residences, including the dogs positive for VL, and forest areas., Conclusions/significance: The EPA, which is inhabited by humans, has an active VL focus. The risk of establishing and maintaining disease transmission foci in similar scenarios, i.e. wild areas that undergo environmental modifications, is evident. Moreover, different epidemiological profiles of VL must be included to elaborate prevention and control measures that consider the particularities of each transmission area.

Bats have aroused growing attention in the public health sphere because they are considered the main reservoir of rabies virus (RABV) in the Americas, in places where canine rabies is under control. Antigenic and genetic studies of RABV isolates have been used to describe the epidemiological profile of rabies and to identify possible hosts/reservoirs for different epidemiological cycles. This study describes the antigenic and genotypic characterization of 19 RABV isolates from central nervous system samples of non-hematophagous bats (Mammalia: Chiroptera). These bats were diagnosed as RABV positive by direct fluorescent antibody and mouse inoculation tests. Antigenic characterization using a panel of eight monoclonal antibodies revealed that 7 of 19 RABV isolates from these bats belonged to variant 3, for which the hematophagous bat species Desmodus rotundus is the main reservoir, and 1 of 19 RABV isolates from an insectivorous bat belonged to variant 4, which is characteristic of these bats. The remaining 11 RABV samples were divided into six non-compatible profiles. The isolates were subjected to reverse transcription polymerase chain reaction for the N gene and partially sequenced. Genetic characterization of these isolates was performed by grouping the sequences obtained with known RABV lineages. The sequences were grouped in clusters by the phylogenetic inference neighbor-joining method, together with another 89 homologous sequences obtained from GenBank. This analysis grouped the isolates into four known lineages: Nyctinomops Brazil, Myotis Brazil, Eptesicus Brazil and D. rotundus Brazil, as well as another cluster that may define a RABV lineage not yet characterized, here named Myotis Brazil II, for which bats of the genus Myotis apparently act as reservoirs. This assumption of a new lineage is also based on the observation of amino acid substitutions, with an average intraspecific identity of 99.8%, varying from 99.6 to 100.0% for nucleotides and 100.0% for amino acids.

Cows are probably the main source of contamination of raw milk with Staphylococcus aureus. Mammary glands with subclinical mastitis can shed large numbers of Staph. aureus in milk. Because of the risk of this pathogen to human health as well as animal health, the aim of this paper was to describe an outbreak of mastitis caused by methicillin-resistant Staph. aureus (MRSA), oxacillin-susceptible mecA-positive Staph. aureus (OS-MRSA), and methicillin-susceptible Staph. aureus (MSSA) on a dairy farm. Milk samples were obtained from all quarters, showing an elevated somatic cell count by the California Mastitis Test. The isolates were identified by phenotypic and genotypic methods. Staphylococcus spp. were isolated from 53% (61/115) of the milk samples, with 60 isolates identified as Staph. aureus (98.4%) and 1 isolate identified as Staphylococcus epidermidis (1.6%). The presence of the mecA gene was verified in 48.3% of Staph. aureus isolates. Of the Staph. aureus isolates, 23.3% were MRSA and 25.0% were OS-MRSA. The total of mastitis cases infected with MRSA was 12.2%. The detection of this large percentage of mastitis cases caused by MRSA and OS-MRSA is of great concern for the animals' health, because β-lactams are still the most important antimicrobials used to treat mastitis. In addition, Staph. aureus isolates causing bovine mastitis represent a public health risk., (Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

There is a growing concern about the participation of wild hosts and reservoirs in the epidemiology of leishmaniasis, particularly within the context of increasingly frequent environmental changes and the expansion of the One Health concept. This work is a molecular research of infection by Leishmania spp. among the wildlife of an environmentally protected area located in the municipality of Campinas, São Paulo, Brazil. The studied area has a history of intense environmental changes, with notifications of human cases of cutaneous leishmaniasis in the 1990s, and a focus of canine visceral leishmaniasis since 2009. Eighty-two wild mammals were sampled by monthly captures in this region over a 1-year period. Blood samples were collected from each animal and subjected to DNA extraction and PCR using primers for the region of the internal transcribed spacer-1. The results of gene sequencing for the first time revealed the infection of opossums (Didelphis albiventris) by Leishmania spp., subgenera Leishmania and Viannia, in Campinas. These findings, in addition to environmental and historical characteristics of the studied area, indicate a possible role of wildlife in the introduction and/or maintenance of natural foci of leishmaniasis transmission.

Introduction:: Road-killed wild animals host zoonotic pathogens such as Toxoplasma gondii, offering a new opportunity for the epidemiological study of these infectious organisms., Methods: This investigation aimed to determine the presence of T. gondii and other apicomplexan parasites in tissue samples of 64 road-killed wild animals, using polymerase chain reaction (PCR). Positive samples were then typed by PCR-restriction fragment length polymorphism (RFLP) using 7 markers: SAG1, 5'-3'SAG2, SAG3, BTUB, c29-6, PK1, and Apico. PCR-RFLP targeting 18S ribosomal RNA (rRNA) genes was also performed on all samples to detect other apicomplexan parasites., Results: T. gondii DNA was detected in 16 tissue samples from 8 individual animals, as follows: 1 Cerdocyon thous (crab-eating fox), 1 Didelphis albiventris (white-eared opossum), 1 Lutreolina crassicaudata (lutrine opossum), 2 Myrmecophaga tridactyla (giant anteater), 1 Procyon cancrivorus (crab-eating raccoon), and 2 Sphiggurus spinosus (Paraguay hairy dwarf porcupine). Seven different T. gondii genotypes were identified, 6 of which were novel. Typing by 18S rRNA verified these 16 T. gondii-infected samples, and identified 1 Sarcocystis spp.-infected animal [Dasypus novemcinctus (nine-banded armadillo)]. The amplified T. gondii (GenBank accession No. L37415.1) and Sarcocystis spp. 18S rRNA products were confirmed by sequencing., Conclusions: Our results indicate that T. gondii is commonly present in wild mammals, which act as sources of infection for humans and animals, including other wild species. The approach employed herein proved useful for detecting T. gondii and Sarcocystis spp. in the environment and identifying their natural reservoirs, contributing to our understanding of host-parasite interactions.

Toxoplasma gondii is one of the most significant parasite, due its importance in veterinary medicine and in public health, considered a food-borne pathogens, there is no available drug treatments to eliminate it from animal tissue, this reinforce the search for a vaccine against this parasite. This study was aimed to evaluate the dynamic of the distribution of T. gondii in tissues of female Wistar rats and their milk, after the immunization by oral rote with irradiated tachyzoites. One week after pregnancy confirmation, rats was challenged by gavage with T. gondii bradyzoites, oocysts or tachyzoites of T. gondii. Forty-eight pregnant rats were grouped as follows: immunized and challenged with bradyzoites (BZ*); non-immunized and challenged with bradyzoites (BZ); immunized and challenged with oocysts (OC*); non-immunized and challenged with oocysts (OC); immunized and challenged with tachyzoites (TZ*); non-immunized and challenged with tachyzoites (TZ); only immunized (I); control group (C). After parturition, milk samples were collected for 3 weeks and then rats were sacrificed and the tissues and milk samples were researched for T. gondii parasite load determined by the quantitative PCR (qPCR). It was verified that the immunization with irradiated tachyzoites of T. gondii induced the reduction of parasitic load in muscle samples in rats challenged by bradyzoites and oocysts, although not enabled the development of sterile immunity. The detection of parasite DNA in milk was found throughout the lactation period, from immunized and non-immunized rats, however no differences were found in the parasite load caused by immunization., (Copyright © 2015. Published by Elsevier Inc.)

Toxoplasmosis, caused by an obligate intracellular protozoan parasite, Toxoplasma gondii, is an worldwide parasitic disease, with significant importance for animal production and considerable impact to the public health. This study was aimed to evaluate the dynamic of the distribution of T.gondii in tissues of female Wistar rats and their puppies tissues, after the immunization by oral rote with irradiated tachyzoites. One week after pregnancy confirmation, rats was challenged by gavage with T. gondii bradyzoites, oocysts or tachyzoites of T. gondii. Forty-eight pregnant rats were grouped as follow: immunized and challenged with bradyzoites (BZ*); non-immunized and challenged with bradyzoites (BZ); immunized and challenged with oocysts (OC*); non-immunized and challenged with oocysts (OC); immunized and challenged with tachyzoites (TZ*); non-immunized and challenged with tachyzoites (TZ); only immunized (I); control group (C). After parturition the rats were sacrificed and the tissues were researched for the DNA of T. gondii by polymerase chain reaction (PCR) and the parasite load determined by the quantitative PCR (qPCR). It was verified that the immunization with irradiated tachyzoites of T. gondii induced the reduction of parasitic load in most organs analyzed, although not prevent the establishment of infection with the parasite. And also, the immunization showed a favorable effect on the birth rate and litter size., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Road-killed wild animals have been classified as sentinels for detecting such zoonotic pathogens as Leishmania spp., offering new opportunities for epidemiological studies of this infection., Methods: This study aimed to evaluate the presence of Leishmania spp. and Leishmania chagasi DNA by PCR in tissue samples (lung, liver, spleen, kidney, heart, mesenteric lymph node and adrenal gland) from 70 road-killed wild animals., Results: DNA was detected in tissues of one Cavia aperea (Brazilian guinea pig), five Cerdocyon thous (crab-eating fox), one Dasypus septemcinctus (seven-banded armadillo), two Didelphis albiventris (white-eared opossum), one Hydrochoerus hydrochoeris (capybara), two Myrmecophaga tridactyla (giant anteater), one Procyon cancrivorus (crab-eating raccoon), two Sphiggurus spinosus (porcupine) and one Tamandua tetradactyla (lesser anteater) from different locations in the Central Western part of São Paulo state. The Leishmania chagasi DNA were confirmed in mesenteric lymph node of one Cerdocyon thous. Results indicated common infection in wild animals., Conclusions: The approach employed herein proved useful for detecting the environmental occurrence of Leishmania spp. and L. chagasi, as well as determining natural wild reservoirs and contributing to understand the host-parasite interaction.

Background: With the aim of studying Leptospira spp. infection in sheep herds, blood samples and respective kidney and liver fragments were collected from 100 animals from twenty different properties during slaughter at a meat company in the Sorocaba region, São Paulo state, southeast Brazil. The microscopic agglutination test (MAT) was performed with 29 strains of Leptospira spp. To identify the agent in the liver and kidney, 100 samples of each tissue were submitted to culture in Fletcher medium and analyzed by the polymerase chain reaction (PCR) for Leptospira spp., Results: MAT detected 23 samples serologically positive for one or more Leptospira spp. serovars and significantly more for Autumnalis. Eight (4%) samples were positive in culture (four kidneys and four livers), corresponding to five animals with positive serology (one animal simultaneously positive for both kidney and liver) and two negatives. PCR detected Leptospira spp. in 14 samples (seven kidneys and seven livers) corresponding to 12 positive animals (two animals simultaneously positive for kidney and liver), of which ten were serologically positive and two negative., Conclusions: PCR was faster, more practical and more sensitive than culture for detecting leptospires. The results reinforce the importance of sheep in the epidemiological context of leptospirosis.

The objective of this study was to isolate and identify the main staphylococcal species causing bovine mastitis in 10 Brazilian dairy herds and study their capability to produce enterotoxins. Herds were selected based on size and use of milking technology, and farms were visited once during the study. All mammary glands of all lactating cows were screened using the California Mastitis Test (CMT) and a strip cup. A single aseptic milk sample (20 mL) was collected from all CMT-positive quarters. Identification of Staphylococcus spp. was performed using conventional microbiology, and PCR was used to determine the presence of enterotoxin-encoding genes (sea, seb, sec, and sed). Of the 1,318 CMT-positive milk samples, Staphylococcus spp. were isolated from 263 (19.9%). Of these isolates, 135 (51%) were coagulase-positive staphylococci (CPS) and 128 (49%) were coagulase-negative staphylococci (CNS). Eighteen different species of CNS were isolated, among which S. warneri, S. epidermidis and S. hyicus were the most frequent. The distribution of Staphylococcus species was different among herds: S. epidermidis was found in 8 herds, S. warneri was found in 7 herds, and S. hyicus in 6 herds. Some of the CNS species (S. saprophyticus ssp. saprophyticus, S. auricularis, S. capitis, and S. chromogenes) were isolated in only one of the farms. Genes related to production of enterotoxins were found in 66% (n=85) of all CNS and in 35% of the CPS isolates. For both CNS and CPS isolates, the most frequently identified enterotoxin genes were sea, seb, and sec; the prevalence of sea differed between CPS (9.5%) and CNS (35.1%) isolates. Staphylococcus warneri isolates showed a greater percentage of sea than seb, sec, or sed, whereas S. hyicus isolates showed a greater percentage of sea than sec. Over 60% of CNS belonged to 3 major species, which carried 62.2 to 81.3% of the enterotoxin genes. The high prevalence highlights the potential for food poisoning caused by these species. For possible high-risk situations for food poisoning, such as milk produced with total bacterial counts greater than regulatory levels and stored under inappropriate temperatures, monitoring contamination with CNS could be important to protect human health. Because the prevalence of CNS intramammary infections in dairy herds is usually high, and these species can be found in great numbers in bulk milk, identification of risk factors for production of staphylococcal enterotoxins should be considered in future studies., (Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Leptospirosis is an infectious disease of worldwide importance. The development of diagnostic techniques allows sick animals to be identified, reservoirs to be eliminated and the disease prevented and controlled. The present study aimed to compare different techniques for diagnosing leptospirosis in sheep. Samples of kidney, liver and blood were collected from 465 animals that originated from a slaughterhouse. The sera were analyzed by the Microscopic Agglutination Test (MAT), and kidney and liver samples of seropositive animals were analyzed using four techniques: bacteriological culture, the Warthin Starry (WS) technique, conventional PCR (cPCR), and quantitative PCR (qPCR). With the MAT, 21 animals were positive (4.5%) to serovars Hardjo (n=12), Hebdomadis (n=5), Sentot (n=2), Wolfii (n=1) and Shermani (n=1). Titers were 100 (n=10), 200 (n=2), 400 (n=6) and 1600 (n=3). No animal was positive by bacteriological culture; four animals were positive by the WS technique in kidney samples; six animals were positive by cPCR in kidney samples; and 11 animals were positive by qPCR, eight of which in kidney samples and three in liver. The bacterial quantification revealed a median of 4.3 bacteria/μL in liver samples and 36.6 bacteria/μL in kidney samples. qPCR presented the highest sensitivity among the techniques, followed by cPCR, the WS technique and bacteriological culture. These results indicate that sheep can carry leptospires of the Sejroe serogroup, and demonstrate the efficiency of quantitative PCR to detect Leptospira spp. in tissue samples., (Published by Elsevier B.V.)

White piedra is a superficial mycosis caused by the genus Trichosporon. It is characterized by nodules on the hair shaft. Pediculosis capitis is caused by Pediculus humanus var. capitis of the suborder Anoplura. Whereas pediculosis is a common infestation, clinical reports of white piedra are rare. Molecular biology procedures identified T. inkin as the agent of white piedra in this case report. The authors present associations between the two diseases in the same patient in order to highlight their clinical differences.

Introduction: White piedra is a superficial mycosis caused by the genus Trichosporon and characterized by nodules on hair shaft., Methods: The authors report a family referred to as pediculosis. Mycological culture on Mycosel® plus molecular identification was performed to precisely identify the etiology., Results: A Trichosporon spp. infection was revealed. The molecular procedure identified the agent as Trichosporon inkin., Conclusions: White piedra and infection caused by T. inkin are rarely reported in Southern Brazil. The molecular tools are essentials on identifying the Trichosporon species.

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.

Toxoplasmosis is the major parasitic disease affecting sheep. It is important for veterinary medicine, animal science and public health since it causes reproductive and economic losses in the herd, as well as damaging human health due to consumption of contaminated meat and milk, which can facilitate zoonotic transmission. Detection of Toxoplasma gondii in ovine milk and lack of data in the literature describing differentiation between acute and chronic disease for this species stimulated the elaboration of the present research project. To achieve the aim of this study, the animals were allocated to two groups of 20 ewes each, of which group 1 was composed of animals with positive serology and group 2 with negative serology. Acute and chronic stages of the disease were differentiated by modified direct agglutination test (MAT), in which antigens were fixed with formalin (MAT-AF) and methanol (MAT-AM). The parasite was detected in milk by polymerase chain reaction (PCR), and the molecular identity of the amplified products was confirmed by sequencing. The serological results indicated that sheep had a chronic infection profile. T. gondii DNA was detected in seven milk samples from five seropositive sheep, and twice in milk of two sheep. Sequences of species shared 97-100% identity with T. gondii. These findings allowed the hypothesis that the peripartum period may also lead to the resurgence of tissue T. gondii tachyzoites cysts which can circulate again and be excreted in the milk. This study used sheep naturally infected with T. gondii as a prerequisite for further investigations on the possible participation of this species in toxoplasmosis epidemiology and as a potential transmission route related to consumption of milk from infected sheep., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The aim of this work was to molecularly detect Leishmania species in 52 cats from Andradina Municipality, São Paulo State, Brazil. The direct parasitological test was performed by using imprints of poplited lymph node, bone marrow and spleen to verify amastigote forms of Leishmania spp. The samples that were positive parasitological tests were subjected to molecular analysis (PCR) and sequencing. Infection was detected for 5.76% (3/52) of the examined cats and two had presence of amastigote forms of Leishmania spp. in lymph nodes. Polymerase chain reaction (PCR) of kinetoplast minicircle DNA, indicated positive amplification for samples of spleen and lymph nodes and the sequencing resulted in 97% similarity with Leishmania (L.) chagasi. This study proved the occurrence of infection with Leishmania (L.) chagasi in felines from Andradina municipality, São Paulo State., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Synanthropic rodents, mainly rats and mice, become ecologically associated with men due to changes in their ecosystems caused by human activities. These animals may take part in the epidemiological cycles of several diseases, including toxoplasmosis. The presence of serum antibodies to Toxoplasma gondii in 43 rodents captured in the urban area of Umuarama, PR, Brazil, was verified by modified agglutination test (MAT). Brain and heart samples were also collected and bioassayed in mice for the isolation of the parasite. Isolated samples were analyzed by 12 multilocus genotyping. Although all rodents were seronegative, the parasite was isolated in one mouse (Mus musculus) and one rat (Rattus rattus). Genotyping showed that these samples were similar to those previously isolated from cats in the state of Parana, Brazil., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background: Several pathogens that cause important zoonotic diseases have been frequently associated with armadillos and other xenarthrans. This mammal group typically has evolved on the South American continent and many of its extant species are seriously threatened with extinction. Natural infection of armadillos with Paracoccidioides brasiliensis in hyperendemic areas has provided a valuable opportunity for understanding the role of this mammal in the eco-epidemiology of Paracoccidioidomycosis (PCM), one of the most important systemic mycoses in Latin America., Findings: This study aimed to detect P. brasiliensis in different xenarthran species (Dasypus novemcinctus, Cabassous spp., Euphractus sexcinctus, Tamandua tetradactyla and Myrmecophaga tridactyla), by molecular and mycological approaches, in samples obtained by one of the following strategies: i) from road-killed animals (n = 6); ii) from naturally dead animals (n = 8); iii) from animals that died in captivity (n = 9); and iv) from living animals captured from the wild (n = 2). Specific P. brasiliensis DNA was detected in several organs among 7/20 nine-banded armadillos (D. novemcinctus) and in 2/2 anteaters (M. tridactyla). The fungus was also cultured in tissue samples from one of two armadillos captured from the wild., Conclusion: Members of the Xenarthra Order, especially armadillos, have some characteristics, including a weak cellular immune response and low body temperature, which make them suitable models for studying host-pathogen interaction. P. brasiliensis infection in wild animals, from PCM endemic areas, may be more common than initially postulated and reinforces the use of these animals as sentinels for the pathogen in the environment.

Medical mycology has greatly benefited from the introduction of molecular techniques. New knowledge on molecular genetics has provided both theoretical and practical frameworks, permitting important advances in our understanding of several aspects of pathogenic fungi. Considering Paracoccidioides brasiliensis in particular, important eco-epidemiological aspects, such as environmental distribution and new hosts were clarified through molecular approaches. These methodologies also contributed to a better understanding about the genetic variability of this pathogen; thus, P. brasiliensis is now assumed to represent a species complex. The present review focuses on some recent findings about the current taxonomic status of P. brasiliensis, its phylogenetic and speciation processes, as well as on some practical applications for the molecular detection of this pathogen in environmental and clinical materials.

Paracoccidioides brasiliensis infections have been little studied in wild and/or domestic animals, which may represent an important indicator of the presence of the pathogen in nature. Road-killed wild animals have been used for surveillance of vectors of zoonotic pathogens and may offer new opportunities for eco-epidemiological studies of paracoccidiodomycosis (PCM). The presence of P. brasiliensis infection was evaluated by Nested-PCR in tissue samples collected from 19 road-killed animals; 3 Cavia aperea (guinea pig), 5 Cerdocyon thous (crab-eating-fox), 1 Dasypus novemcinctus (nine-banded armadillo), 1 Dasypus septemcinctus (seven-banded armadillo), 2 Didelphis albiventris (white-eared opossum), 1 Eira barbara (tayra), 2 Gallictis vittata (grison), 2 Procyon cancrivorus (raccoon) and 2 Sphiggurus spinosus (porcupine). Specific P. brasiliensis amplicons were detected in (a) several organs of the two armadillos and one guinea pig, (b) the lung and liver of the porcupine, and (c) the lungs of raccoons and grisons. P. brasiliensis infection in wild animals from endemic areas might be more common than initially postulated. Molecular techniques can be used for detecting new hosts and mapping 'hot spot' areas of PCM.

Background: Paracoccidioides brasiliensis ecology is not completely understood, although several pieces of evidence point to the soil as its most probable habitat. The present study aimed to investigate the fungal growth, conidia production and molecular pathogen detection in different soil conditions., Methods: Soils samples of clayey, sandy and medium textures were collected from ground surface and the interior of armadillo burrows in a hyperendemic area of Paracoccidioidomycosis. P. brasiliensis was inoculated in soil with controlled humidity and in culture medium containing soil extracts. The molecular detection was carried out by Nested PCR, using panfungal and species specific primers from the ITS-5.8S rDNA region., Results: The soil texture does not affect fungus development and the growth is more abundant on/in soil saturated with water. Some soil samples inhibited the development of P. brasiliensis, especially those that contain high values of Exchangeable Aluminum (H+Al) in their composition. Some isolates produced a large number of conidia, mainly in soil-extract agar medium. The molecular detection was positive only in samples collected from armadillo burrows, both in sandy and clayey soil., Conclusion: P. brasiliensis may grow and produce the infectious conidia in sandy and clayey soil, containing high water content, mainly in wild animal burrows, but without high values of H+Al.