Equidae pythiosis in Brazil and the world: a systematic review of the last 63 years (1960-2023).

This systematic review compiles reports of clinical pythiosis in horses, mules and donkeys from 1960 to 2023 worldwide, focusing on Brazil. We searched databases and included 71 articles detailing clinical characteristics, geographic distribution, epidemiology, diagnostic methods, therapies, and outcomes. The results showed that publications on equine pythiosis have significantly increased since 2010. Brazil reported the highest incidence, comprising 55% of cases, predominantly in the southern, northeastern, and central-western regions during summer and autumn. Cutaneous pythiosis was the most prevalent form, generally presenting as single lesions in the appendicular region, and affected females more than males. Diagnosis typically involved histopathology, used alone or with other methods. Various treatments have been employed, with surgery, often combined with chemotherapy and immunotherapy, being the most common. Notably, 80.84% of treated animals recovered, highlighting the effectiveness of these therapies in enhancing survival rates. The limitations of the study included the lack of data in published case reports, which made it difficult to collect and calculate epidemiological data. Additionally, we recognize that pythiosis in Brazil is underreported, since this disease does not have mandatory notification and several cases are not registered and/or reported in the literature. Lastly, it is hypothesized that equid pythiosis may be more widespread than currently known, and its real occurrence in Brazil remains uncertain.

In vitro and ex vivo anti-Pythium insidiosum potential of ozonated sunflower oil.

This study sought to evaluate the in vitro and ex vivo susceptibility of Pythium insidiosum to ozonized sunflower oil (OSO) and verify the morphological alterations of OSO-exposed hyphae. Susceptibility assays were performed according to the broth microdilution protocol M38-A2/CLSI, and the minimal inhibitory (MIC) and minimal oomicidal (MOC) concentrations were also determined. Non-ozonated sunflower oil (SO) was used as the oil control. Additionally, kunkers from equine pythiosis were exposed to OSO. Damages caused by OSO and SO on P. insidiosum hyphae ultrastructure were verified using scanning electron microscopy. The MIC range for OSO was 7000 to 437.5 mg/mL, and the values for SO were higher, ranging from 56000 to 14000 mg/mL. The MOC was equal to MIC for both oil formulations. The OSO fully inhibited the oomycete growth from kunkers, although there was P. insidiosum growth in the kunker control in 24 h of incubation. The SEM analyses showed that both OSO and SO caused morphological alterations in P. insidiosum hyphae, highlighting the presence of cavitation along the hyphae with loss of continuity of the cell wall, which was more evident in the OSO-treated hyphae. The OSO had the best oomicidal activity, leading us to believe that our findings may support future research containing this formulation to be applied in integrative medicine protocols to control pythiosis in animals and humans.

Oomicidal activity of polypyrrole nanoparticles against Pythium insidiosum.

This study evaluated in-vitro action of a new molecule, the polypyrrole nanoparticles (Ppy-NP), against Pythium insidiosum isolates using M38-A2/CLSI; the minimal inhibitory (MIC) and minimal oomicidal (MOC) concentrations were also determined. Additionally, changes in the hyphae wall of P. insidiosum CBS 575.85 treated with Ppy-NP were evaluated by scanning electron microscopy (SEM). The MIC100 and MOC for all isolates ranged from 8 to 32 µg mL-1, and the MIC90 and MIC50 were 16 µg mL-1. The SEM showed structural damage to the hyphae of P. insidisoum treated with Ppy-NP, as hyphae surfaces with less turgidity were found, thereby showing scaling and ruptures compared to the control (untreated hyphae). Our findings highlighted the anti-P. insidiosum properties of Ppy-NP proved to be a promising candidate for research using pythiosis experimental models.

In vitro lytic efficiency of Staphylococcus aureus bacteriophages in bacteria from bovine mastitis: a meta-analysis / Eficiência lítica in vitro de bacteriófagos de Staphylococcus aureus em bactérias de mastites bovina: uma meta-análise

ABSTRACT: Bacteriophages have been investigated as alternative to the treatment of bacterial infections, including bovine mastitis, in production animals. In this meta-analysis, we evaluated in vitro efficiency of phages of Staphylococcus aureus against S. aureus, which is involved in the etiology of bovine mastitis. Seventeen studies were included and the bacterial lytic activity was extracted using proportion analysis. The lytic efficiency of phages was obtained in this meta-analysis using a random-effects model [significant difference (P<0.05)]. Forest plots were used to graphically represent the efficiency of phages on bacterial isolates. Most phages (e.g., CS1, DW2, ΦSA011, ΦSA012, ΦSA022, ΦSA023, ΦSA024, ΦSA025, ΦSA037, ΦSA038, ΦSA039, ΦSA041, ΦSA042, ΦSA043, ΦSA044, MSA6, Ufv-aur2 to Ufv-aur11, SAH-1, SPW, vB_SauM_JS25, SaPh1 to SaPh6, SA, SANF, SA2, ΦSA012, ΦSA039, phi11, phiIPLA88, phiIPLA35, phiIPLA-RODI, phiIPLA-C1C, SAJK-IND, vBSP-A1, vBSP-A2, STA1.ST29, EB1.ST11, EB1.ST27, Remus, and ISP) were efficiently lytics or infected most S. aureus isolates, demonstrating 80% (P<0.05) lytic efficiency. The phages SA, SANF and SA2, also demonstrated lytic activity or infected the non-Staphylococcus aureus and Macrococcus caseolyticus isolates. In this meta-analysis, we compared and demonstrated the in vitro efficiency and host range of S. aureus phages. Additionally, the phages represent an alternative to be researched to treat bovine mastitis in dairy cattle caused by the prevalent microorganism, S. aureus.

RESUMO: Os bacteriófagos têm sido investigados como alternativa ao tratamento de infecções bacterianas em animais de produção, incluindo a mastite bovina. Nesta meta-análise, avaliamos a eficiência in vitro de fagos de Staphylococcus aureus contra S. aureus envolvidas na etiologia da mastite bovina. Dezessete estudos foram incluídos e a atividade lítica bacteriana foi extraída usando análise de proporção. A eficiência lítica dos fagos foi obtida nesta meta-análise, usando um modelo de efeitos aleatórios (diferença significativa (P <0,05)). Os gráficos de Forest plots foram usados para representar graficamente a eficiência dos fagos em isolados bacterianos. Os fagos avaliados, na sua grande maioria, (por exemplo, CS1, DW2, ΦSA011, ΦSA012, ΦSA022, ΦSA023, ΦSA024, ΦSA025, ΦSA037, ΦSA038, ΦSA039, ΦSA041, ΦSA042, ΦSA043, ΦSAf e U0f, ua04 SPW, vB_SauM_JS25, SaPh1 a SaPh6, SA, SANF, SA2, ΦSA012, ΦSA039, phi11, phiIPLA88, phiIPLA35, phiIPLA-RODI, phiIPLA-C1C, SAJK-IND, vBSP-A1, vBSP-A2, STA1.ST29, EB1.ST11, EB1.ST27, Remus, e ISP) foram eficientemente líticos ou infectaram a maioria dos isolados de S. aureus, demonstrando 80% (P < 0,05) de eficiência lítica. Os fagos SA, SANF e SA2 também demonstraram atividade lítica ou infectaram os isolados Staphylococcus não-aureus e Macrococcus caseolyticus. Nesta meta-análise, comparamos e demonstramos a eficiência in vitro e gama de hospedeiros de fagos de S. aureus. Adicionalmente, os fagos representam uma alternativa a ser pesquisada para o tratamento da mastite bovina em gado leiteiro causada pelo microrganismo prevalente, ou seja S. aureus.

Origanum vulgare essential oil: antibacterial activities and synergistic effect with polymyxin B against multidrug-resistant Acinetobacter baumannii.

Antimicrobial resistance is increasing around the world and the search for effective treatment options, such as new antibiotics and combination therapy is urgently needed. The present study evaluates oregano essential oil (OEO) antibacterial activities against reference and multidrug-resistant clinical isolates of Acinetobacter baumannii (Ab-MDR). Additionally, the combination of the OEO and polymyxin B was evaluated against Ab-MDR. Ten clinical isolates were characterized at the species level through multiplex polymerase chain reaction (PCR) for the gyrB and blaOXA-51-like genes. The isolates were resistant to at least four different classes of antimicrobial agents, namely, aminoglycosides, cephems, carbapenems, and fluoroquinolones. All isolates were metallo-ß-lactamase (MßL) and carbapenemase producers. The major component of OEO was found to be carvacrol (71.0%) followed by ß-caryophyllene (4.0%), γ-terpinene (4.5%), p-cymene (3,5%), and thymol (3.0%). OEO showed antibacterial effect against all Ab-MDR tested, with minimum inhibitory concentrations (MIC) ranging from 1.75 to 3.50 mg mL-1. Flow cytometry demonstrated that the OEO causes destabilization and rupture of the bacterial cell membrane resulting in apoptosis of A. baumannii cells (p < 0.05). Synergic interaction between OEO and polymyxin B (FICI: 0.18 to 0.37) was observed, using a checkerboard assay. When combined, OEO presented until 16-fold reduction of the polymyxin B MIC. The results presented here indicate that the OEO used alone or in combination with polymyxin B in the treatment of Ab-MDR infections is promising. To the best of our knowledge, this is the first report of OEO and polymyxin B association against Ab-MDR clinical isolates.

In vitro algicidal effect of polypyrrole on Prototheca species isolates from bovine mastitisAlgicidal activity of polypyrrole on Prototheca spp.

Algae of the genus Prototheca are microorganisms involved in the occurrence of diseases in humans and animals. In bovine species, Prototheca spp. cause environmental mastitis, productive losses in dairy herds, mainly leading to the discard of infected cows. Currently, there are no effective anti-Prototheca spp. drugs to combat this infection. Thus, the search for an efficacious therapy for Prototheca spp. infections have become essential. Highly soluble polypyrrole (Ppy) is a molecule with known antimicrobial activity. This study aimed to characterize Prototheca spp. isolates from bovine mastitis as well as to evaluate the susceptibility profile and to verify the morphological alterations on Prototheca spp. isolates treated with Ppy. In this research, 36 Brazilian isolates of Prototheca spp. were characterized by restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR) assay for the mitochondrial cytB gene. Additionally, Ppy algicidal activity against these isolates of Prototheca spp. was assessed by minimal microbicidal concentration method in microplates. Further, scanning electron microscopy (SEM) was performed in order to verify the morphological alterations on Prototheca spp. isolates in response to Ppy. The isolates were characterized as belonging to Prototheca zopfii genotype 2 (35/36) and Prototheca blaschkeae (1/36). Ppy had an algicidal effect on all isolates tested at concentrations ranging from 15.625 µg ml-1 to 62.5 µg ml-1. SEM showed changes on planktonic and sessile P. zopfii, including a decrease of the number of cells with the presence of an amorphous substance involving the cells. The algicidal activity of Ppy suggests the therapeutic potential of this molecule in the prevention and treatment of Prototheca spp. in bovine mastitis.

Biogenic silver nanoparticles in the treatment of experimental pythiosis Bio-AgNP in pythiosis therapy.

Pythiosis is a rapidly progressing disease that can be lethal to affected individuals due to resistance to available therapeutic protocols. The disease affects mammals, with the largest number of reports in horses and humans. The present study investigated the activity of biogenic silver nanoparticles (bioAgNP) in the treatment of experimental pythiosis. The disease was reproduced in nine female 90-day-old New Zealand rabbits. Animals were divided into three groups: group1 (control, n = 3) daily and topically treated with a nonionized gel-based formulation and 1 ml of sterile distilled water intralesion administered every 48 hours; group 2 (n = 3), daily and topically treated with gel-based formulation containing 1 µg/ml bio-AgNP; group 3 (n = 3), treated with 1 ml bio-AgNP in 1 µg/ml aqueous solution intralesion administered every 48 hours. Animals were treated for 45 days, and the area of subcutaneous lesions was measured every 5 days. Results showed that groups 2 and 3 differed from control group (P < .05) in the lesion area, as well as the amount of hyphae within the lesions. It was observed that lesions of treated animals (groups 2 and 3) did not differ from each other, showing that the application route did not influence the regression of lesions. However, it was observed that one animal from group 2 reached clinical cure at 35 days of treatment. This research is pioneer in the application of nanocomposites for the treatment of experimental pythiosis and showed that bio-AgNP can be powerful allies of integrative medicine and can be included in pythiosis therapeutic protocols.

Exposure of Toxocara canis eggs to Purpureocillium lilacinum as a biocontrol strategy: an experimental model evaluation / Exposição de ovos de Toxocara canis a Purpureocillium lilacinum como estratégia de biocontrole: uma avaliação em modelo experimental

Abstract Purpureocillium lilacinum is a nematophagous fungus used in biological control against some parasites, including Toxocara canis. This study researched the infectivity of embryonated T. canis eggs after exposure to the fungus P. lilacinum. T. canis eggs were exposed to P. lilacinum for 15 or 30 days and subsequently administered to Swiss mice (n=20). Control group consisted of mice who received T. canis embryonated eggs without fungal exposure. Forty-eight hours after infection, heart, lung, and liver from animals of each group were collected to assess larval recovery. The organs of mice that received embryonated eggs exposed to the fungus showed a lower average larval recovery (P<0.05) suggesting that exposure of T. canis eggs to P. lilacinum was able to reduce experimental infection. Under the evaluated conditions, the interaction time between the fungus and the parasite eggs was not a significant factor in larvae recovery. P. lilacinum may be considered a promising T. canis biological control agent. However, further studies are needed to determine a protocol for the use of this fungus as a biological control agent.

Resumo Purpureocillium lilacinum é um fungo nematófago com potencial para uso no controle biológico de parasitos, incluindo Toxocara canis. Este estudo pesquisou a infectividade de ovos de T. canis embrionados após exposição ao fungo P. lilacinum . Ovos de T. canis foram expostos ao fungo por 15 ou 30 dias e subsequentemente administrados a camundongos Swiss (n=20). O grupo controle consistiu de camundongos que receberam ovos embrionados do parasita sem exposição ao fungo. Quarenta e oito horas após a infecção, coração, pulmão e fígado dos camundongos foram coletados para avaliar a recuperação larval. Os órgãos dos animais que receberam ovos embrionados expostos ao fungo apresentaram menor média de recuperação larval (P<0,05) do que os infectados com ovos sem exposição ao fungo, sugerindo que a exposição dos ovos de T. canis a P. lilacinum foi capaz de reduzir a infecção experimental. Nas condições avaliadas, o tempo de interação entre o fungo e os ovos do parasito não foi um fator significativo na recuperação das larvas. P. lilacinum pode ser considerado um promissor agente de controle biológico de T. canis, no entanto, mais estudos são necessários para avaliar o emprego deste fungo como um agente de controle biológico.

Genotyping of South American clinical isolates of Pythium insidiosum based on single nucleotide polymorphism-based multiplex PCR / Genotipagem de isolados clínicos de Pythium insidiosum da América do Sul utilizando polimorfismos de nucleotídeo único baseado em PCR multiplex

ABSTRACT: We aimed to genotype the South American clinical isolates of Pythium insidiosum using the single nucleotide polymorphisms (SNP) of the ribosomal DNA sequences (rDNA). Previously, an SNP-based multiplex-PCR was able to distinguish three different clades of P. insidiosum isolates. Thus, we used this assay to evaluate South American clinical isolates of P. insidiosum (n=32), standard strains from Costa Rica (n=4), Thailand (n=3), Japan (n=1), and India (n=1), a standard strain of Pythium aphanidermatum, and Brazilian environmental isolates of Pythium torulosum, Pythium rhizo-oryzae and Pythium pachycaule voucher (n=3). It was possible to allocate each American P. insidiosum isolate to clade I, the isolates of India, Japan, and Thailand to clade II, and the Thai isolate to clade III. P. aphanidermatum, P.torulosum, P.rhizo-oryzae and P.pachycaule voucher isolates were not amplified. For the first time, a P. insidiosum isolate from Uruguay, South America, was included in molecular analyzes. By SNP-based multiplex-PCR, it was possible to perform the identification and genotyping of the South American isolates of P. insidiosum, demonstrating similar genetic characteristics of these isolates.

RESUMO: O objetivo deste estudo foi genotipar isolados clínicos de Pythium insidiosum da América do Sul utilizando polimorfismos de nucleotídeo único (SNP) de sequências de rDNA. Anteriormente, um multiplex-PCR baseado em SNP foi capaz de distinguir P. insidiosum em três diferentes clados. Dessa forma, utilizamos este método para avaliar isolados clínicos de P. insidiosum da América do Sul (n=32), cepas padrão da Costa Rica (n=4), Tailândia (n=3), Japão (n=1) e Índia (n=1), uma cepa padrão de Pythium aphanidermatum e isolados ambientais brasileiros de Pythium torulosum; Pythium rhizo-oryzae e Pythium pachycaule voucher (n=3). Os isolados analisados foram alocados aos clados: I (americanos), II (isolados da Índia, Japão e Tailândia), e III (um isolado tailandês). P. aphanidermatum, P.torulosum, P.rhizo-oryzae e P.pachycaule voucher não foram amplificados. Pela primeira vez, um isolado de P. insidiosum do Uruguai foi incluído em análises moleculares. Através da multiplex-PCR baseada em SNP, foi possível realizar a identificação e genotipagem dos isolados sul-americanos de P. insidiosum, demonstrando características genéticas semelhantes entre esses isolados.

Risk factors and infection due to Cryptosporidium spp. in dogs and cats in southern Rio Grande do Sul / Fatores de risco e infecção por Cryptosporidium spp. em cães e gatos no sul do Rio Grande do Sul

Abstract This study investigated the frequency of oocysts of Cryptosporidium spp. in feces from dogs and cats in five municipalities in the southern region of the state of Rio Grande do Sul. The risk factors associated with infection were also investigated. Feces samples from 110 dogs and 18 cats were stained using the auramine method. At the time of feces sampling, a questionnaire with semi-open-ended questions was applied to the animal guardians and all data obtained underwent statistical analysis. The real frequency of oocysts of Cryptosporidium spp. was 24.63% (27 dogs and two cats). Only four samples of dog feces were diarrheic and no presence of oocysts was observed in any of them. Variables that represented risk factors for infection were: homemade food, untreated water, circulation of animals on grassy terrain and living in the same environment as other animals (cattle). The results made it possible to inferring that within the population studied, the frequency of parasitism due to Cryptosporidium spp. in dogs was relevant and emphasize the asymptomatic nature of this infection. The adopting control measures are highlighted, particularly in relation to variables that represent risk factors for this infection.

Resumo Este estudo verificou a frequência de oocistos de Cryptosporidium spp. em fezes de cães e gatos em cinco municípios da região sul do Rio Grande do Sul e fatores de risco associados à infecção. Amostras de fezes de 110 cães e 18 gatos foram coradas pelo método de auramina. No momento da coleta de fezes aplicou-se um questionário aos tutores dos animais com questões semiabertas e os dados foram submetidos à análise estatística. A frequência verdadeira de oocistos de Cryptosporidium spp. foi de 24,63% (27 cães e 2 gatos). Apenas quatro amostras de fezes caninas eram diarreicas e todas sem oocistos. As variáveis que representaram fatores de risco para a infecção foram: alimentos de preparo caseiro, água não tratada, circulação dos animais em terreno gramíneo e convivência com outros animais, principalmente bovinos. Os resultados sugerem que a frequência de cães parasitados por Cryptosporidium spp. é relevante, reforçando o caráter assintomático da infeção. Destaca-se a importância da adoção de medidas de controle, particularmente das variáveis que representaram fatores de risco à infecção.

Isolation of Saprolegnia aenigmatica oomycetes and protocol for experimental infection of pacu (Piaractus mesopotamicus) / Isolamento do oomiceto Saprolegnia aenigmatica e protocolo de infecção experimental em pacu (Piaractus mesopotamicus)

Successful disease treatment depends on molecular studies under indoor conditions with experimental infection protocols that facilitate understanding the disease and the drug`s efficacy. The internal transcribed spacer (ITS) region was sequenced from three isolates, which were identified as Saprolegnia aenigmatica. Subsequently, healthy fish were immunosuppressed with dexamethasone (1.2 mg kg-1) and descaled to the skin using a sharp scalpel. These individuals were isolated in individual aquariums maintained at 22°C. Individuals in one group were subcutaneously inoculated with 9,000 zoospores (DDZ treatment), a second group was exposed to oomycetes in water with three colonized baits (DDB), a third group was maintained in water without zoospores (DD), and a control group (C) consisted of healthy animals. After 48 and 96 hours, two animals from each group were euthanized for fungal reisolation. The fish from groups DD and C did not show clinical signs, and no oomycetes were isolated. The animals from the DDZ and DDB groups showed cotton-wool-like masses on the skin, and S. aenigmatica was re-isolated. Thus, for infection using zoospores or baits parasitized by S. aenigmatica, an immunosuppressor (dexamethasone) and a sharp scalpel can be used effectively to establish an experimental infection in P. mesopotamicus.

O sucesso do tratamento de uma enfermidade depende do estudo de moléculas em condições de laboratório por meio de protocolos de infecção experimental que viabilizam a compreensão da doença e da eficácia dos fármacos. Pela sequência ITS foram identificadas três cepas de Saprolegnia aenigmatica. Dessa forma, pacus sadios foram submetidos à imunossupressão com dexametasona (1,2 mg kg-1), esfoliados com auxílio de bisturi e distribuídos em aquários a 22ºC. Após este procedimento, um grupo de animais foi inoculado com 9.000 zoósporos/peixe subcutâneo (DEZ), a outro foram adicionadas três iscas colonizadas com o oomiceto na água (DEI), um terceiro grupo foi mantido sem contato com o oomiceto (DE) e um quarto grupo, de animais sadios, representaram o controle (C). Após 48 e 96h deste procedimento, foram eutanaziados animais de cada grupo para reisolamento. Os animais do grupo DE e C não apresentaram sinais clínicos e não foi reisolado o oomiceto. Porém, tanto os animais do grupo DEZ quanto como os animais do grupo DEI apresentaram micélio branco na pele e foi reisolado Saprolegnia aenigmatica. Assim, a infecção com zoósporos ou com iscas colonizadas por S. aenigmatica, com o uso de dexametasona e abrasivo epitelial são formas eficazes de infecção experimental em P. mesopotamicus.

In vitro susceptibility of the oomycete Pythium insidiosum to metallic compounds containing cadmium, lead, copper, manganese or zinc.

Pythium insidiosum is an aquatic oomycete that causes pythiosis, an important and severe disease of difficult treatment that affects humans, domestic and wild animals. This infection is often described in horses in Brazil and humans in Thailand. In clinical practice, we have observed many cases that do not respond to available therapies, indicating the need to explore alternative therapeutic approaches. In this sense, studies using metal compounds in conjunction with available antimicrobial agents have been demonstrated greater antimicrobial activity. Thus, in this research, we tested in vitro activities of metallic compounds containing cadmium, lead, copper, manganese, or zinc against 23 isolates of P. insidiosum. The assays were performed by broth microdilution based on CLSI M38-A2 document. The minimum inhibitory and fungicidal concentrations were established for all isolates. Copper acetate and cadmium acetate showed the highest inhibitory effects, with minimal inhibitory concentration ranging from 4-64 µg/ml and 16-256 µg/ml, respectively. The mean geometric for minimal fungicidal concentrations were, respectively, 26 µg/ml and 111.43 µg/ml for copper acetate and cadmium acetate. These results suggest that copper and cadmium can inhibit P. insidiosum growth, highlighting the greater inhibitory activity of copper acetate. In addition, our results propose that copper and/or cadmium compounds can be used in upcoming researches to formulate effective new complexed drugs against P. insidiosum in in vitro and in vivo experimental models.

Effect of anaerobic bovine colostrum fermentation on bacteria growth inhibition / Efeito da fermentação anaeróbica do colostro bovino na inibição do crescimento de bactérias

ABSTRACT: Efficient handling programs that provide high quality colostrum in adequate amounts to dairy farm calves are needed to assure their health and survival. Replacers (or milk substitutes) often become necessary when colostrum presents inadequate quality, or in order to break the cycle of infectious disease transmission. In this study we aimed to assess the effect of anaerobic fermentation processing (colostrum silage) on bacterial that represent interest to animal health. Colostrum samples were inoculated with cultures of Brucella abortus , Escherichia coli , Leptospira interrogans serovar Copenhageni , Mycobacterium bovis , Salmonella Enteritidis , Salmonella Typhimurium , Staphylococcus aureus , and Bacillus cereus and then subjected to anaerobic fermentation. On the first day, and every seven days until 30th days after fermentation, the samples were cultured and colony forming units counted. At seven days of fermentation, B. abortus , L. interrogans , and M. bovis were not detected. At 14th days of fermentation, E. coli , S. aureus , S. Enteritidis and S. Typhimurium were no longer detected. However, we were able to detect both lactic acid bacteria and B. cereus until 30th days of fermentation. From this study we suggested that anaerobic fermentation processing can inhibit important bacteria that cause economical losses for the cattle industry. The observations suggested that colostrum silage is a promising form to conserve bovine colostrum.

RESUMO: Eficientes programas de manejo que permitem o fornecimento de colostro de alta qualidade e em quantidades adequadas para bezerros são necessários para garantir a sua saúde e sobrevivência. Substitutos do leite tornam-se frequentemente necessários quando o colostro apresenta qualidade inadequada ou a fim de quebrar o ciclo de transmissão de doenças infecciosas. Este estudo teve como objetivo avaliar o efeito da fermentação anaeróbica do colostro bovino (silagem de colostro) sobre bactérias de interesse para a saúde animal. Alíquotas de colostro foram inoculadas com culturas de Brucella abortus , Escherichia coli , Leptospira interrogans serovar Copenhageni , Mycobacterium bovis , Salmonella Enteritidis , Salmonella Typhimurium , Staphylococcus aureus e Bacillus cereus e submetidas ao processo de fermentação anaeróbia. No primeiro dia e a cada sete dias até o 30o dia de fermentação, as alíquotas foram cultivadas e analisadas pela contagem de unidades formadoras de colônias. Aos sete dias de fermentação, unidades formadoras de colônias de B. abortus, L. interrogans e M. bovis não foram detectadas e unidades formadoras de colônia de E. coli , S. aureus , S. Enteritidis e S. Typhimurium não foram observadas no 14o dia de fermentação. No entanto, B. cereus e bactérias ácido lácticas foram detectadas até o 30o dia de fermentação. Este estudo sugere que o processo de fermentação anaeróbia do colostro bovino proporciona efeitos inibitórios sobre as bactérias avaliadas, constituindo-se num promissor método de conservação do colostro bovino.

In Vitro Activity of Melaleuca alternifolia (Tea Tree) in Its Free Oil and Nanoemulsion Formulations Against Pythium insidiosum.

Pythium insidiosum is an important aquatic oomycete which can cause pythiosis in both animals and humans. This microorganism shows low susceptibility to antifungal drugs available. This study analyzed the in vitro antimicrobial activity of Melaleuca alternifolia in its free oil (FO) and nanoemulsion (NE) formulations against Brazilian P. insidiosum isolates. The antimicrobial activity evaluation was performed by the broth microdilution method according to CSLI M38-A2 document adapted to phytopharmaceuticals. Twenty-six P. insidiosum isolates were evaluated, and the minimum inhibitory concentration was determined at 100 % growth inhibition. Melaleuca alternifolia essential oil or FO was obtained commercially. The NE containing 1 % M. alternifolia essential oil was prepared by the spontaneous emulsification method. All P. insidiosum isolates evaluated showed minimum inhibitory concentrations (MIC) ranging from 531.5 to 2125 µg/mL for the FO formulation; MIC50 and MIC90 showed values between 1062.5 and 2125 µg/mL, respectively. When the NE formulation was evaluated, MIC values ranged from 132.7 to 2125 µg/mL and both MIC50 and MIC90 corresponded to 1062.5 µg/mL. FO and NE formulations of M. alternifolia showed antimicrobial activity against P. insidiosum. This study demonstrated that M. alternifolia oil can be an additional therapy in pythiosis treatment; however, further studies are needed to evaluate the applicability of the plant essential oils in the treatment of clinical pythiosis.

Epidemiological, clinical and diagnostic aspects of sheep conidiobolomycosis in Brazil / Aspectos epidemiológicos, clínicos e de diagnóstico da conidiobolomicose ovina no Brasil

ABSTRACT: Conidiobolomycosis is an emerging disease caused by fungi of the cosmopolitan genus Conidiobolus. Particular strains of Conidiobolus coronatus, Conidiobolus incongruus and Conidiobolus lamprauges, mainly from tropical or sub-tropical origin, cause the mycosis in humans and animals, domestic or wild. Lesions are usually granulomatous and necrotic in character, presenting two clinical forms: rhinofacial and nasopharyngeal. This review includes the main features of the disease in sheep, with an emphasis on the epidemiology, clinical aspects, and diagnosis of infections caused by Conidiobolus spp. in Brazil. In this country, the disease is endemic in the Northeast and Midwest, affecting predominantly woolless sheep breeds and occasioning death in the majority of the studied cases. The species responsible for infections of sheep are C. coronatus and C. lamprauges and the predominant clinical presentation is nasopharyngeal. These fungal infections are very important, since they compromise the health status of the sheep flock and cause serious economic losses to the sheep industry. Thus, research is needed to investigate faster tools for diagnosis and effective methods for the control and treatment of conidiobolomycosis.

RESUMO: Conidiobolomicose é uma enfermidade emergente causada por fungos cosmopolitas do gênero Conidiobolus. Isolados de Conidiobolus coronatus, Conidiobolus incongruus e Conidiobolus lamprauges, principalmente de origem tropical e subtropical, afetam humanos, animais domésticos e silvestres. As lesões da conidiobolomicose normalmente são de caráter granulomatoso e necrótico, apresentando-se sob duas formas clínicas: rinofacial e nasofaríngea. O presente artigo tem como objetivo revisar as principais características da doença em ovinos, particularizando a epidemiologia, assim como os aspectos clínicos e o diagnóstico das infecções causadas por Conidiobolus spp. no Brasil. Neste País, a enfermidade é endêmica nas regiões nordeste e centro-oeste, afetando ovinos predominantemente de raças deslanadas, ocasionando a morte na grande maioria dos casos estudados. As espécies do fungo responsáveis pelas infecções em ovinos são C. coronatus e C. lamprauges e a forma clínica predominante é a nasofaríngea. Tais infecções fúngicas são muito importantes, uma vez que comprometem o status sanitário do rebanho ovino e acarretam sérios prejuízos econômicos à ovinocultura. Dessa forma, pesquisas são necessárias para investigar ferramentas rápidas de diagnóstico e métodos eficazes de controle e tratamento da conidiobolomicose.

Isolation and molecular characterization of symbiotic fungus from Acromyrmex ambiguus and Acromyrmex heyeri ants of Rio Grande do Sul State, Brazil / Isolamento e caracterização molecular do fungo simbionte de formigas-cortadeiras (Acromyrmex heyeri e Acromyrmex ambiguus) do Rio Grande do Sul, Brasil

Leaf-cutting ants of the genera Atta and Acromyrmex determine serious agricultural problems and live on symbiosis with Leucoagaricus gongylophorus. The aim of this study is to identify morphological and molecularly, as well as to verify the genotypic variability of the symbiotic fungus cultivated by A. heyeri and A. ambiguus from three different regions of the state of Rio Grande do Sul, Brazil. Fungus gardens were collected and fragments of mycelia were grown in selective medium. Total DNA was extracted and amplification of the ITS region was performed by PCR using universal primers. After DNA sequencing, the chromatograms were assembled and phylogenetic analyzes were performed by the Neighbor-Joining method. A total of six isolates of L. gongylophorus were obtained and their identities were confirmed by molecular analyses. Phylogenetic analysis of the ITS region showed a tree with two distinct groups regarding the fungus isolates from A. heiyeri and A. ambiguous. In this study, it was verified that A. heyeri and A. ambiguous, cultivate the same fungus. Additionally, the molecular marker used in this study showed variations in L. gongylophorus, evidencing two distinct branches in the phylogenetic tree, according to the ant species that cultivate L. gongylophorus. However, other studies involving the inclusion of a great number of isolates of L. gongylophorus, as well as the use of other molecular markers to validate the possible variations in the phylogenetic relationship of this symbiotic fungus are required.

Formigas-cortadeiras dos gêneros Atta e Acromyrmex causam elevados prejuízos à agricultura e são dependentes obrigatórias da simbiose com Leucoagaricus gongylophorus. O objetivo deste estudo foi identificar morfologicamente e molecularmente, bem como verificar a variabilidade genotípica do fungo simbionte,cultivado por Acromyrmex heyeri e Acromyrmex ambiguus em três regiões do RS, Brasil. Jardins de fungo foram coletados do interior de formigueiros e fragmentos de micélio foram cultivados em meio de cultura seletivo. O DNA total foi extraído e a amplificação da região ITS foi realizada por PCR, utilizando primers universais. Após sequenciamento, os cromatogramas foram montados e as análises filogenéticas foram realizadas pelo método de Neighbor-Joining. Dos jardins de fungo, obtiveram-se seis isolados de L. gongylophorus, confirmados por análise molecular. A análise filogenética da região ITS mostrou uma árvore com dois grupos distintosem relação aos isolados do fungo oriundos de ninhos de A. heyeri e A. ambiguus. Neste estudo, evidenciou-se que as espécies de formigas A. heyeri e A. ambiguus cultivam o mesmo fungo. Entretanto, o marcador molecular estudado evidenciou variações de L. gongylophorus que permitiram formar duas ramificações diferentes na árvore filogenética relacionada à espécie de formiga que o cultiva. Todavia, para validar as possíveis variações nas relações filogenéticas deste fungo simbionte, é necessária a inclusão de um maior número de isolados de L. gongylophorus, bem como o emprego de outros marcadores moleculares.

Anaerobically fermented colostrum: an alternative for feeding calves / Colostro fermentado anaerobicamente: uma alternativa para alimentação de bezerras

Milk or commercial milk replacer blends are the most expensive components in final costs of calves breeding. Colostrum is available and it is the appropriate sources for calves' nutrition, being an excellent option as milk substitute. Besides having both nutritional and immunological characteristics that are superior to milk, colostrum represents no costs to the producer. However, difficulties in preservation of colostrum generate controversy results. The aim of this study was to evaluate the anaerobically fermented colostrum (colostrum silage) as liquid diet for dairy calves. We evaluated the microbiological and physicochemical properties of silage, and performance of 31 animals up to 60 days age. From 21 days until 360 days of fermentation we isolated only bacteria of the genus Lactobacillus spp. The physicochemical evaluation of colostrum silage revealed a tendency to maintain the protein, dry matter and fat values during the evaluation period. The average weight gain of calves fed with milk was 0.6kg day-1 for female and 0.6g day-1 for males while those fed with colostrum silage was significantly higher (P<0.05) with a mean of 0.7kg day-1 for female and 0.7kg day-1 for males. The results of this study demonstrated that colostrum silage keeps the properties necessary for the development of the calves and is a suitable replacement for calves' milk.

O leite ou substitutos comerciais do leite são os componentes mais caros do custo final da criação de bezerras. O colostro é uma fonte adequadada de nutrientes, sendo uma excelente opção como substituto do leite. Além de ter características nutricionais e imunológicas superiores ao leite, o colostro não representa custos para o produtor. No entanto, as dificuldades de preservação do colostro têm gerado resultados controversos. O objetivo deste estudo foi avaliar o colostro fermentado anaerobicamente (silagem de colostro) como dieta líquida para bezerras leiteiras. Foram avaliadas as propriedades microbiológicas e físico-químicas da silagem de colostro e o desempenho de 31 animais até 60 dias de idade. A partir de 21 dias até 360 dias de fermentação, foram isoladas apenas bactérias do género Lactobacillus spp. A avaliação físico-química da silagem de colostro revelou uma tendência para manter os valores de proteína, gordura e matéria seca durante o período de avaliação. O ganho médio de peso dos bezerros alimentados com leite foi de 0,6kg dia-1 para as fêmeas e de 0.6kg dia-1 para os machos, enquanto que o ganho médio de peso para os animais alimentados com silagem colostro foi significativamente maior (P<0,05), com média de 0,7kg dia-1 para as fêmeas e 0,7kg dia-1 para os machos. Os resultados deste estudo mostram que a silagem de colostro mantém as propriedades necessárias para o desenvolvimento dos animais, sendo um substituto adequado para o leite.

Efeito in vitro de químicos no crescimento micelial de Saprolegnia spp. / In vitro effect of chemical on hyphal growth of Saprolegnia spp.

Com o aumento da produtividade na piscicultura, os peixes são expostos a altas densidades de estocagem que podem levar ao estresse e imunossupressão. Essa condição favorece a ocorrência de infecções, entre elas a saprolegniose que afeta os peixes e seus ovos, causando sérios prejuízos econômicos aos piscicultores. Dessa forma, compostos químicos eficazes e "ambientalmente amigáveis" são almejados para o controle da doença. O objetivo do presente estudo foi avaliar a suscetibilidade in vitro de 12 isolados de Saprolegnia spp., provenientes dos peixes-rei dulceaquícolas Odontesthes bonariensis e O. humensis. Para isso, testes de suscetibilidade, avaliando a inibição do crescimento micelial de Saprolegnia spp., foram realizados frente a seis químicos (cloreto de sódio, formaldeído, permanganato de potássio, iodopovidona, sal marinho e sal marinho iodado) nas concentrações seriadas de 0 a 10.000ppm. Os resultados obtidos evidenciaram que o formaldeído e o permanganato de potássio, em concentrações a partir de 10 e acima de 100ppm, respectivamente, inibiram o crescimento micelial de isolados Saprolegnia spp.. Já a iodopovidona e os compostos salinos não evidenciaram atividade antimicrobiana em nenhuma das concentrações testadas (0-10.000ppm). Embora os resultados in vitro demonstrem que formaldeído e permanganato de potássio sejam promissores no controle da saprolegniose, estudos futuros deverão avaliar a eficácia e o efeito in vivo desses compostos nos peixes-rei O. bonariensis e O. humensis.

The productivity in fish farming has been increasing and consequently the fish are submitted to high stocking densities which usually cause stress and immunosuppression. These conditions determine the occurrence of a series of infections, including saprolegniosis. This disease affects fish and eggs from freshwater determining relevant economic losses to fish farmers. Thus, to avoid outbreaks, are being sought effective chemical agents that are "environmentally friendly". The aim of this study was to evaluate the in vitro susceptibility of 12 isolates of Saprolegnia spp. from the silversides Odontesthes bonariensis and O. humensis. Susceptibility tests evaluating inhibition of mycelial growth of Saprolegnia spp. were performed using six chemicals (sodium chloride, formaldehyde, potassium permanganate, povidone-iodine, sea salt and iodized sea salt) in concentrations from 0 to 10,000ppm. The results displayed that formaldehyde at the concentration of 10ppm, and potassium permanganate at concentrations above 100ppm, were able to inhibit the mycelial growth of isolates of Saprolegnia spp. However, povidone-iodine and saline compounds exhibited no antimicrobial activity on Saprolegnia spp. Although the in vitro results showed that formaldehyde and potassium permanganate could be promising in controlling saprolegniosis, further studies should be performed to evaluate the in vivo efficacy of these compounds as well as to verify the toxicity of the chemicals to the silversides O. bonariensis and O. humensis.

Perfuração gástrica em um canino induzida por nimesulida / Gastric perforation in a canine induced by nimesulide

Os anti-inflamatórios não esteroides (AINEs) têm sido muito utilizados na clínica de pequenos animais (FOX e JOHNSTON, 1997). Esses medicamentos possuem excelentes efeitos terapêuticos no alívio da dor e no tratamento de processos inflamatórios, no entanto possuem efeitos adversos especialmente no trato gastrointestinal onde podem promover o aparecimento de úlceras focais. As úlceras são lesões caracterizadas pela destruição da mucosa do trato gastroentérico (WILLARD, 2003). O objetivo deste trabalho é relatar o tratamento cirúrgico de um canino diagnosticado com úlcera gástrica perfurante devido ao uso de nimesulida.

Princípios de biossegurança aplicados aos laboratórios de ensino universitário de microbiologia e parasitologia / Principles of biosafety applied to microbiology and parasitology laboratories in universities

Os laboratórios de ensino de microbiologia e parasitologia nas universidades brasileiras são ambientes em que as atividades integradas, envolvendo ensino, pesquisa e extensão, demandam a convivência de pessoas, agentes e amostras biológicas, equipamentos, reagentes e resíduos num mesmo espaço, sendo inevitável a exposição das pessoas aos diferentes riscos. As atividades realizadas nesses laboratórios necessitam empregar as normas de segurança; pois, uma vez que o fator humano é susceptível aos acidentes, os programas de educação em biossegurança são imprescindíveis. Entretanto, para que esses programas sejam efetivos, é necessário que os usuários estejam devidamente informados acerca dos princípios de biossegurança, bem como aptos a colocá-los em prática de maneira correta, a fim de garantir a segurança dos profissionais, acadêmicos e do meio ambiente. Esta revisão compila os principais aspectos da biossegurança relacionados aos princípios e à classificação dos riscos, dos agentes biológicos e dos níveis de contenção laboratorial, bem como aborda as boas práticas laboratoriais nos laboratórios de ensino, pesquisa e extensão em microbiologia e parasitologia.

The teaching laboratories of microbiology and parasitology in Brazilian universities are places where the integrated activities of teaching, research and extension are performed and require the coexistence in the same space, persons, biological agents and samples, equipment, reagents and waste, and where is inevitable the exposure of people to the different risks. The laboratorial activities practiced in Brazilian universities require the need for safety standards, since the human factor is likely the causes of accidents in laboratories, educational programs in biosafety become indispensable to the prevention of risks. However, for an education program become effective is necessary that the users are fully informed about the biosafety principles, as well as able to practice them properly to ensure the safety of all professionals, academics and the environment. This article compiles the main aspects related to the principles of biosafety, risk classification of biological agents and levels of biocontainment, and addresses the issues regarding safety equipment and good laboratory practices applied to the teaching laboratories in microbiology and parasitology.