Potential in vitro action of an adenosine analog and synergism with penicillin against Corynebacterium pseudotuberculosis.

Caseous lymphadenitis is a well-known disease caused by Corynebacterium pseudotuberculosis affecting small ruminants with small significance to human health because of its minor zoonotic potential. In both cases, few treatment options are available and conventional antimicrobial therapy is commonly refractory due to development of pyogranulomatous reactions, bringing great interest in discovering novel therapeutics for more suitable approaches. Dideoxynucleotides presented antibacterial action against various bacteria but were never described for C. pseudotuberculosis. Hypothesizing the antimicrobial action of 2',3'-dideoxiadenosine (ddATP) against C. pseudotuberculosis, we performed for the first time an investigation of its minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) in the ATCC® 19,410 strain and a well-characterized clinical isolate of C. pseudotuberculosis. We also assessed potential synergism with penicillin. ddATP showed a growth delay effect for C. pseudotuberculosis at 2 µmol/mL and a MIC and MBC of 4 µmol/mL against the ATCC® 19,410 strain, but not for the clinical strain. An antimicrobial effect was observed when using concentrations lower than the MIC of ddATP associated with penicillin for both strains tested. Our data suggest the potential of nucleotide analogs, especially adenosine, and its combination with penicillin, as a possible novel treatment for C. pseudotuberculosis-induced infections, and contributes with knowledge regarding alternative drugs to treat C. pseudotuberculosis infections.

Bacteriological, cytological, and molecular investigation of Corynebacterium pseudotuberculosis, mycobacteria, and other bacteria in caseous lymphadenitis and healthy lymph nodes of slaughtered sheep.

Caseous lymphadenitis (CL) in sheep is a chronic contagious disease caused by Corynebacterium pseudotuberculosis, commonly characterized by abscess formation in peripheral lymph nodes and disseminated infections. Nonetheless, other microorganisms, including with zoonotic relevance, can be isolated from CL-resembling lymph nodes. Currently, mycobacteria have been reported in visceral granulomatous lesions in small ruminants, a fact that poses a public health issue, particularly in slaughtered sheep intended for human consumption. Cytology using fine needle aspiration and microbiological culturing are suitable tests for routine diagnostic, whereas present drawbacks and molecular methods have been confirmatory. Data about the occurrence of mycobacteria in both lymph nodes with aspect of CL and apparently healthy visceral nodes of sheep slaughtered for human consumption are scarce. In this study, 197 visceral lymph nodes of sheep showed lymphadenitis and 202 healthy visceral lymph nodes of slaughtered sheep intended for human consumption were submitted to conventional bacteriological diagnosis, mycobacteria culturing, and cytological evaluation. Compatible Corynebacterium isolates were subjected to multiplex PCR targeting 16S rRNA, rpoB, and pld genes to detect C. pseudotuberculosis. Based on microbiological identification, C. pseudotuberculosis (86/197; 43.7%), streptococci γ-hemolytic (17/197; 8.6%), and Trueperella pyogenes (12/197; 6.1%) were prevalent in lymph nodes with abscesses, as opposed to staphylococci (53/202; 26.2%) in apparently healthy lymph nodes. No mycobacteria were isolated. Cytology identified 49.2% (97/197) Gram-positive pleomorphic organisms (coryneform aspect). Multiplex PCR confirmed genetic material of C. pseudotuberculosis in 74.4% (64/86) of the samples with C. pseudotuberculosis isolation and 66% (64/97) samples with cytological coryneform aspect (κ = 86.78%; 95% CI = 79.87-93.68%). These findings emphasize the prevalence of C. pseudotuberculosis in abscess formation among peripheral lymph nodes of sheep. Other bacteria were also identified in lymph nodes sampled that resembling C. pseudotuberculosis-induced infections that may difficult the diagnosis. Multiplex PCR revealed a valuable assay to detect C. pseudotuberculosis, in addition to routine methods applied to CL-diagnosis. No mycobacteria were identified in lymph nodes sampled, with and without apparent lesions. Nonetheless, due to public health impacts, this pathogen should be considered as a differential diagnosis of C. pseudotuberculosis-induced infections during inspection procedures of slaughtered sheep intended for human consumption.

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.

Biofilm formation by Prototheca zopfii isolated from clinical and subclinical bovine mastitis in distinct growth conditions under different dyes / Formação de biofilme por Prototheca zopfii isolados de mastite bovina clínica e subclínica em distintas condições de crescimento sob diferentes corantes

ABSTRACT: Prototheca spp. have been reported as an emergent environmental mastitis pathogen in several countries. Biofilm formation is a significant factor associated with different degrees of virulence developed by many microorganisms, including Prototheca spp. The present study aimed to compare two growth conditions and two staining dyes to determine which combination was more appropriate to evaluate qualitatively and quantitatively the production of biofilm by P. zopfii. Biofilm formation was evaluated in polystyrene microplates under static and dynamic growth conditions and staining with crystal violet or cotton blue dye. All P. zopfii isolates from cows with mastitis were classified as biofilm-producers in all growth conditions and staining. The cotton blue dye proved to be more appropriate method to classify the intensity of P. zopfii biofilm production.

RESUMO: Prototheca spp. tem sido relatado como um patógeno ambiental causador de mastite bovina em vários países. A formação de biofilme é um fator associado a diferentes graus de virulência desenvolvidos por muitos microrganismos, incluindo Prototheca zopfii. O presente estudo teve como objetivo comparar duas condições de crescimento e dois corantes para determinar a combinação mais adequada para avaliar qualitativa e quantitativamente a produção de biofilme por P. zopfii. A formação de biofilme foi avaliada em microplacas de poliestireno sob condições estáticas e dinâmicas de crescimento e coloração com cristal violeta ou azul de algodão. Todos os isolados de P. zopfii de vacas com mastite foram caracterizados como produtores de biofilme, independentemente das condições de crescimento e coloração. O corante azul de algodão demonstrou ser o método mais adequado para classificar a intensidade de produção de biofilme de P. zopfii.

Anti-influenza A virus antibodies in Tayassuidae from commercial rearing farms in Brazil / Detecção de anticorpos contra vírus influenza A em Tayassuidae de criações comerciais no Brasil

ABSTRACT: Family Tayassuidae in the suborder Suina include two species of peccaries in Brazil: the white-lipped peccary (Tayassu pecari) and the collared peccary (Pecari tajacu). These animals share common pathogens with domestic swine (Sus scrofa); however, their role as potential carrier remains unclear. This study focused on detecting the prevalence of influenza A antibodies in Tayassu pecari and Pecari tajacu from commercial rearing farms from two states in Brazil. A set of 50 blood samples from Pecari tajacu and 55 from Tayassu pecari were analyzed using a commercial indirect ELISA in order to investigate anti influenza A antibodies. Pecari tajacu samples presented 22% (11/50) of seropositivity for the virus. Serological surveillance is an important tool to identify the presence and the spread of the influenza virus in feral pigs.

RESUMO: A família Tayassuidae pertencente a subordem Suina e compreende duas espécies presentes no Brasil: Queixada (Tayassu pecari) e o Caititu (Pecari tajacu). Ambas as espécies compartilham patógenos com o suíno doméstico (Sus scrofa), entretanto o papel destes animais como carreadores destas infecções permanece indefinido. O presente estudo teve como objetivo detectar a ocorrência de anticorpos contra vírus influenza A em amostras de soro de rebanhos comerciais de queixada e caititu, provenientes de dois estados do Brasil. Um total de 50 amostras de soro de Pecari tajacu e 55 amostras de Tayassu pecari foram testadas por meio de ELISA, sendo que 22% (11/50) das amostras de Pecari tajacu foram soropositivas para o agente. Estudos de vigilância sorológica são importantes para identificar a presença e a disseminação do vírus influenza em suínos selvagens.

Multidrug resistant bacteria isolated from septic arthritis in horses / Bactérias multirresistentes isoladas de artrite séptica equina

Septic arthritis is a debilitating joint infectious disease of equines that requires early diagnosis and immediate therapeutic intervention to prevent degenerative effects on the articular cartilage, as well as loss of athletic ability and work performance of the animals. Few studies have investigated the etiological complexity of this disease, as well as multidrug resistance of isolates. In this study, 60 horses with arthritis had synovial fluid samples aseptically collected, and tested by microbiological culture and in vitro susceptibility test (disk diffusion) using nine antimicrobials belonging to six different pharmacological groups. Bacteria were isolated in 45 (75.0%) samples, as follows: Streptococcus equi subsp. equi (11=18.3%), Escherichia coli (9=15.0%), Staphylococcus aureus (6=10.0%), Streptococcus equi subsp. zooepidemicus (5=8.3%), Staphylococcus intermedius (2=3.3%), Proteus vulgaris (2=3.3%), Trueperella pyogenes (2=3.3%), Pseudomonas aeruginosa (2=3.3%), Klebsiella pneumoniae (1=1.7%), Rhodococcus equi (1=1.7%), Staphylococcus epidermidis (1=1.7%), Klebsiella oxytoca (1=1.7%), Nocardia asteroides (1=1.7%), and Enterobacter cloacae (1=1.7%). Ceftiofur was the most effective drug (>70% efficacy) against the pathogens in the disk diffusion test. In contrast, high resistance rate (>70% resistance) was observed to penicillin (42.2%), enrofloxacin (33.3%), and amikacin (31.2%). Eleven (24.4%) isolates were resistant to three or more different pharmacological groups and were considered multidrug resistant strains. The present study emphasizes the etiological complexity of equine septic arthritis, and highlights the need to institute treatment based on the in vitro susceptibility pattern, due to the multidrug resistance of isolates. According to the available literature, this is the first report in Brazil on the investigation of the etiology. of the septic arthritis in a great number of horses associated with multidrug resistance of the isolates.(AU)

Artrite séptica é uma artropatia infecciosa debilitante de equinos, que requer diagnóstico precoce e intervenção terapêutica imediata, com intuito de evitar a degeneração de a cartilagem articular e a perda da capacidade atlética e de trabalho dos animais. Poucos estudos têm investigado a complexidade etiológica da afecção, bem como a presença de multirresistência dos isolados aos antimicrobianos. Foram investigados 60 equinos portadores de artrite, submetidos à colheita asséptica de líquido sinovial para a realização de cultivo microbiológico e teste de sensibilidade microbiana in vitro (difusão com discos) com nove antimicrobianos pertencentes a seis diferentes grupos farmacológicos. Foi obtido isolamento microbiano em 45 (75,0%) amostras, como segue: Streptococcus equi subsp. equi (11=18,3%), Escherichia coli (9=15,0%), Staphylococcus aureus (6=10,0%), Streptococcus zooepidemicus (5=8,3%), Staphylococcus intermedius (2=3,3%), Proteus vulgaris (2=3,3%), Trueperella pyogenes (2=3,3%), Pseudomonas aeruginosa (2=3,3%), Klebsiella pneumoniae (1=1,7%), Rhodococcus equi (1=1,7%), Staphylococcus epidermidis (1=1,7%), Klebsiella oxytoca (1=1,7%), Nocardia asteroides (1=1,7%) e Enterobacter cloacae (1=1,7%). Ceftiofur foi o antimicrobiano mais efetivo (>70% eficácia) in vitro diante dos patógenos. Em contraste, alta resistência dos isolados (>70% de resistência) foi observada para penicilina (42,2%), enrofloxacino (33,3%) e amicacina (31,2%). Onze (24,4%) isolados foram resistentes a três ou mais diferentes grupamentos de fármacos e considerados com resistência múltipla aos antimicrobianos. O presente estudo enaltece a complexidade etiológica envolvida na artrite séptica em equinos e ressalta a necessidade de instituir o tratamento dos animais com respaldo de testes de sensibilidade microbiana in vitro em virtude da resistência múltipla dos isolados. De acordo com a literatura consultada, esta é a primeira descrição no país da etiologia da artrite séptica em grande número de equinos associada a multirresistência dos isolados aos fármacos testados.(AU)

Successful therapy in unusual generalized Dermatophilus congolensis infection in a calf based on modified in vitro disk diffusion test / Sucesso no tratamento de infecção generalizada em bezerra por Dermatophilus congolensis baseado em teste in vitro modificado de difusão com discos

Bovine dermatophilosis is a dermatitis characterized by typical focal or localized lesions with "paintbrush" aspect and occasionally as disseminated cutaneous disease. We report the case of a one-year-old Nelore female with history of chronic cutaneous disseminated lesions that appeared immediately after a rainfall period. Serous to purulent exudates, hair with tufted appearance, hyperkeratotic, non-pruritic, hardened, yellowish to brown, and coalescent crusty lesions were observed distributed all over its body. Removal of the crusts revealed ulcerated or hemorrhagic areas, with irregular elevated crusts like "paintbrush". Microbiological diagnosis enabled the identification of a microorganism, the Dermatophilus congolensis. Despite disseminated and chronic lesions, we obtained a successful therapy with parenteral therapy using long-acting tetracycline based on modified in vitro disk diffusion test. The present report highlights success therapy in uncommon generalized bovine dermatophilosis with selection of first-choice drugs based on modified in vitro susceptibility test, and need of responsible use of antimicrobials in livestock.(AU)

A dermatofilose bovina é uma dermatite caracterizada por lesões focais ou localizadas com aspecto de "pincel" e, ocasionalmente, como lesão cutânea disseminada. Relata-se o caso de uma fêmea bovina de um ano de idade, que foi atendida apresentando história de lesões cutâneas crônicas imediatamente após um período de alta pluviosidade. Ao exame clínico, lesões serosas a purulentas, com hiperqueratose, coalescentes, não pruriginosas, ressecadas, de coloração amarelada à acinzentada foram observadas distribuídas de modo generalizado pelo animal. A remoção das crostas revelou áreas ulceradas ou hemorrágicas, com crostas irregulares e elevadas semelhantes a "pincel". O diagnóstico microbiológico possibilitou a identificação do micro-organismo Dermatophilus congolensis. Apesar das lesões disseminadas e crônicas, a cura do animal foi obtida com tratamento parenteral usando oxitetraciclina de longa duração, baseado em teste in vitro de sensibilidade microbiana modificado. O presente relato ressalta o sucesso no tratamento de caso incomum de lesões generalizadas de dermatofilose bovina com respaldo de teste in vitro de sensibilidade modificado, bem como a necessidade do uso responsável de antimicrobianos em animais de produção.(AU)

Diagnosis of mycobacteria in bovine milk: an overview

ABSTRACT Tuberculosis remains as the world’s biggest threat. In 2014, human tuberculosis ranked as a major infectious disease by the first time, overcoming HIV death rates. Bovine tuberculosis is a chronic disease of global distribution that affects animals and can be transmitted to humans by the consumption of raw milk, representing a serious public health concern. Despite the efforts of different countries to control and eradicate bovine tuberculosis, the high negative economic impact on meat and milk production chains remains, given the decreased production efficiency (approximately 25%), the high number of condemned carcasses, and increased animal culling rates. This scenario has motivated the establishment of official programs based on regulations and diagnostic procedures. Although Mycobacterium tuberculosis and Mycobacterium bovis are the major pathogenic species to humans and bovines, respectively, nontuberculous mycobacteria within the Mycobacterium genus have become increasingly important in recent decades due to human infections, including the ones that occur in immunocompetent people. Diagnosis of mycobacteria can be performed by microbiological culture from tissue samples (lymph nodes, lungs) and secretions (sputum, milk). In general, these pathogens demand special nutrient requirements for isolation/growth, and the use of selective and rich culture media. Indeed, within these genera, mycobacteria are classified as either fast- or slow-growth microorganisms. Regarding the latter ones, incubation times can vary from 45 to 90 days. Although microbiological culture is still considered the gold standard method for diagnosis, molecular approaches have been increasingly used. We describe here an overview of the diagnosis of Mycobacterium species in bovine milk.

Enterotoxin genes, multidrug resistance, and molecular typing of Staphylococcus spp. isolated from organic bovine milk / Genes de enterotoxinas, multirresistência a antimicrobianos e caracterização molecular de espécies de Staphylococcus spp. isoladas de leite bovino orgânico

The multidrug resistant and the emergence of methicillin-resistant staphylococci isolated from animals, food, and humans are public health concern. These microorganisms produce different toxins related to food poisoning in humans. This study aimed to characterize Staphylococcus spp. isolated from two organic milk farms in Brazil. A total of 259 milk samples were collected, from which 58 (22.4%) Staphylococcus spp. were isolated. The highest sensibility to ceftiofur and sulfamethoxazole/trimethoprim was observed in 96.6% of Staphylococcus spp., and whereas 89% were resistant to penicillin G. The mecA gene was detected in 13.8% of the isolates. SEA and SEC were the most common enterotoxins detected. PFGE revealed genetic heterogeneity from S. intermedius and S. warneri analyzed, while S. aureus presented similar profiles among isolates from the two studied herds. To the best of our knowledge, the current study describes for the first time presence of enterotoxins, mecA gene, and genetic diversity of staphylococci isolated from organic dairy farms in Brazil.(AU)

A emergência de estafilococos multirresistentes e resistentes à meticilina, isolados de animais, alimentos e humanos é uma preocupação em saúde pública. Esses micro-organismos produzem diferentes toxinas relacionadas à intoxicação alimentar em humanos. Este estudo caracterizou Staphylococcus spp. isolados em duas fazendas orgânicas no Brasil. Foram coletadas 259 amostras de leite em duas propriedades leiteiras orgânicas, nas quais 58 (22,4%) estirpes de Staphylococcus spp. foram isoladas. A maior sensibilidade dos isolados foi observada para ceftiofur e sulfametoxazol/trimetoprim em 96,6%. Em contraste, acima de 89% de resistência dos estafilicocos foi encontrada para penicilina G. O gene mecA foi identificado em 13,8% dos isolados. SEA e SEC foram as enterotoxinas mais comumente detectadas. PFGE revelou heterogeneidade genética entre S. intermedius e S. warneri, enquanto S. aureus demonstraram perfis semelhantes entre isolados dos dois rebanhos estudados. Relata-se pela primeira vez no Brasil a detecção de enterotoxinas, o gene mecA e diversidade genética em estafilococos isolados de vacas em produção orgânica.(AU)

Unusual caudal vena cava thrombosis in a cow, secondary to Trueperella (Arcanobacterium) pyogenes infection / Rara trombose da veia cava caudal em bovino, secundária à infecção por Trueperella (Arcanobacterium) pyogenes

The caudal vena cava thrombosis, or pulmonary thromboembolism, in cattle is correlated with lactic acidosis, caused by diets rich in grains and highly fermentable, associated or not to septic situations, used in feedlots of beef or high-producing dairy cattle. This paper reports an unusual caudal vena cava thrombosis in a cow, secondary to Trueperella (Arcanobacterium) pyogenes infection, resulting in reduced milk production, anorexia, pale mucous membranes, ruminal atony, sternal decubitus and autoauscultation position. The heart was enlarged at necropsy, presence of clots distributed along the thoracic cavity, adherence between lung and pleura, abscesses, emphysema, petechiae, suffusions and ecchymosis in lungs, thickening of the caudal vena cava wall, hepatomegaly with chronic passive congestion ("nutmeg" aspect), and rumenitis. In lab, the actinomycete Trueperella (Arcanobacterium) pyogenes was isolated from liver and lung samples, probably resulting through dissemination of the bacteria of the rumen content, what reaffirms the opportunistic behavior of this actinomycete.(AU)

A síndrome da veia cava caudal ou tromboembolismo pulmonar bovino está relacionada à acidose láctica causada por dietas ricas em grãos e altamente fermentáveis, associados ou não a quadros sépticos, usadas em confinamentos de bovinos de corte ou para vacas leiteiras de alta produção. O presente artigo reporta caso raro de trombose da veia cava caudal em uma vaca, secundária a infecção por Trueperella (Arcanobacterium) pyogenes, apresentando reduzida produção de leite, anorexia, palidez de mucosas, atonia ruminal, decúbito esternal e posição de autoauscultação. À necrópsia observou-se coração aumentado de tamanho, coágulos distribuídos por toda cavidade torácica, aderência entre os pulmões e pleura, abscessos, enfisema, petéquias, sufusões, equimoses nos pulmões, espessamento da parede da veia cava caudal com trombo, hepatomegalia com congestão passiva crônica (aspecto de "noz moscada"), e ruminite. Em laboratório isolou-se o actinomiceto Trueperella (Arcanobacterium) pyogenes a partir de amostras de fígado e pulmão, provavelmente resultando da disseminação da bactéria proveniente do conteúdo ruminal, e reafirma o comportamento oportunista deste actinomiceto.(AU)

MOLECULAR IDENTIFICATION AND ANTIMICROBIAL RESISTANCE PATTERN OF SEVEN CLINICAL ISOLATES OF Nocardia spp. IN BRAZIL / Identificação molecular e perfil de sensibilidade a antimicrobianos de sete isolados clínicos de Nocardia spp. no Brasil

Nocardia is a ubiquitous microorganism related to pyogranulomatous infection, which is difficult to treat in humans and animals. The occurrence of the disease is on the rise in many countries due to an increase in immunosuppressive diseases and treatments. This report of cases from Brazil presents the genotypic characterization and the antimicrobial susceptibility pattern using the disk-diffusion method and inhibitory minimal concentration with E-test® strips. In summary, this report focuses on infections in young adult men, of which three cases were cutaneous, two pulmonary, one neurological and one systemic. The pulmonary, neurological and systemic cases were attributed to immunosuppressive diseases or treatments. Sequencing analysis of the 16S rRNA segments (1491 bp) identified four isolates of Nocardia farcinica, two isolates of Nocardia nova and one isolate of Nocardia asiatica. N. farcinica was involved in two cutaneous, one systemic and other pulmonary cases; N. nova was involved in one neurological and one pulmonary case; and Nocardia asiatica in one cutaneous case. The disk-diffusion antimicrobial susceptibility test showed that the most effective antimicrobials were amikacin (100%), amoxicillin/clavulanate (100%), cephalexin (100%) and ceftiofur (100%), while isolates had presented most resistance to gentamicin (43%), sulfamethoxazole/trimethoprim (43%) and ampicillin (29%). However, on the inhibitory minimal concentration test (MIC test), only one of the four isolates of Nocardia farcinica was resistant to sulfamethoxazole/trimethoprim.

Nocardia é um microorganismo ubiquitário relacionado a infecções piogranulomatosas, com difícil resolução tecidual em humanos e animais. A doença é mundialmente emergente devido ao aumento de doenças e tratamentos imunossupressores. Este relato de casos ocorridos no Brasil visa apresentar a identificação molecular dos isolados e o padrão de sensibilidade a antimicrobianos por disco-difusão e concentração inibitória mínima (CIM) através de fitas E-test®. Os casos ocorreram em homens, em idade adulta. Três quadros foram cutâneos, dois pulmonares, um neurológico e um sistêmico. O quadro respiratório, o neurológico e um sistêmico estavam associados à doença ou terapia imunossupressoras. O sequenciamento do gene 16S rRNA (1491pb) possibilitou a identificação de quatro isolados de Nocardia farcinica, dois de Nocardia nova e um de Nocardia asiatica. N. farcinica foi observada em dois casos dermatológicos, um pulmonar e um quadro sistêmico, N. nova foi isolada de um caso neurológico e outro pulmonar; e N. asiatica em um caso dermatológico. O teste de disco-difusão mostrou que amicacina (100%), amoxicilina/clavulanato (100%), cefalexina (100%) e ceftiofur (100%) foram mais efetivos; enquanto gentamicina (43%), sulfametoxazol/trimetoprim (43%) e ampicilina (29%) foram menos efetivos. No entanto, no teste de concentração inibitória mínima (CIM), apenas um dos quatro isolados da espécie Nocardia farcinica mostrou-se resistente a sulfametoxazole-trimetropina.

Deforming mandibular osteomyelitis in a cow caused by Trueperella pyogenes / Osteomielite mandibular deformante em vaca causada por Trueperella pyogenes

This study reports an unusual case of deforming mandibular osteomyelitis in a cow caused by Trueperella (Arcanobacterium) pyogenes, on the face of the ventrolateral caudal portion of the right branch of the mandible. Fragment aspired of lesion by fine needle allowed cytological characterization, isolation and identification of T. pyogenes. Radiographic examination showed marked periosteal reaction in the right mandible, numerous lytic areas and cortical bone destruction. Despite of treatment based on in vitro antimicrobial sensitivity test, it was recommended the euthanasia due to progressive worsening of the cow's condition. Multiple abscesses were observed in the mandibular region at necropsy. Pyogranuloma was characterized in histological exam. Sampled material collected from the lesion after necropsy resulted in microbiological reisolation of T. pyogenes .

Relata-se caso incomum de osteomielite mandibular deformante em vaca, causada por Trueperella (Arcanobacterium) pyogenes, na face ventro-lateral da porção caudal do ramo direito da mandíbula. A punção aspirativa de fragmento da lesão permitiu a caracterização citológica, o isolamento microbiano e identificação de T. pyogenes. Exame radiográfico mostrou acentuada reação periodontal na mandíbula direita, com predomínio de áreas líticas e destruição da cortical óssea. Apesar da instituição do tratamento baseado no teste de sensibilidade microbiana in vitro, foi recomendada a eutanásia, em virtude da piora progressiva do estado geral do animal. No exame post-mortem foram observados múltiplos abscessos na lesão que, histologicamente, foi caracterizada como piogranuloma. A colheita de material da região mandibular afetada, após a necropsia, resultou no reisolamento microbiológico de T. pyogenes .

Molecular identification of nocardia isolates from clinical samples and an overview of human nocardiosis in Brazil.

BACKGROUND: Nocardia sp. causes a variety of clinical presentations. The incidence of nocardiosis varies geographically according to several factors, such as the prevalence of HIV infections, transplants, neoplastic and rheumatic diseases, as well as climate, socio-economic conditions and laboratory procedures for Nocardia detection and identification. In Brazil the paucity of clinical reports of Nocardia infections suggests that this genus may be underestimated as a cause of human diseases and/or either neglected or misidentified in laboratory specimens. Accurate identification of Nocardia species has become increasingly important for clinical and epidemiological investigations. In this study, seven clinical Nocardia isolates were identified by multilocus sequence analysis (MLSA) and their antimicrobial susceptibility was also determined. Most Nocardia isolates were associated to pulmonary disease. METHODOLOGY/PRINCIPAL FINDINGS: The majority of Brazilian human isolates in cases reported in literature were identified as Nocardia sp. Molecular characterization was used for species identification of Nocardia nova, Nocardia cyriacigeorgica, Nocardia asiatica and Nocardia exalbida/gamkensis. Data indicated that molecular analysis provided a different Nocardia speciation than the initial biochemical identification for most Brazilian isolates. All Nocardia isolates showed susceptibility to trimethoprim-sulfamethoxazole, the antimicrobial of choice in the treatment nocardiosis. N. nova isolated from different clinical specimens from one patient showed identical antimicrobial susceptibility patterns and two distinct clones. CONCLUSIONS/SIGNIFICANCE: Although Brazil is the world's fifth-largest country in terms of land mass and population, pulmonary, extrapulmonary and systemic forms of nocardiosis were reported in only 6 of the 26 Brazilian states from 1970 to 2013. A least 33.8% of these 46 cases of nocardiosis proved fatal. Interestingly, coinfection by two clones may occur in patients presenting nocardiosis. Nocardia infection may be more common throughout the Brazilian territory and in other developing tropical countries than is currently recognized and MLSA should be used more extensively as an effective method for Nocardia identification.

Multiplex polymerase chain reaction to identify and determine the toxigenicity of Corynebacterium spp with zoonotic potential and an overview of human and animal infections

Corynebacterium diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis constitute a group of potentially toxigenic microorganisms that are related to different infectious processes in animal and human hosts. Currently, there is a lack of information on the prevalence of disease caused by these pathogens, which is partially due to a reduction in the frequency of routine laboratory testing. In this study, a multiplex polymerase chain reaction (mPCR) assay that can simultaneously identify and determine the toxigenicity of these corynebacterial species with zoonotic potential was developed. This assay uses five primer pairs targeting the following genes: rpoB (Corynebacterium spp), 16S rRNA (C. ulcerans and C. pseudotuberculosis), pld (C. pseudotuberculosis), dtxR (C. diphtheriae) and tox [diphtheria toxin (DT) ]. In addition to describing this assay, we review the literature regarding the diseases caused by these pathogens. Of the 213 coryneform strains tested, the mPCR results for all toxigenic and non-toxigenic strains of C . diphtheriae, C. ulcerans and C. pseudotuberculosis were in 100% agreement with the results of standard biochemical tests and PCR-DT. As an alternative to conventional methods, due to its advantages of specificity and speed, the mPCR assay used in this study may successfully be applied for the diagnosis of human and/or animal diseases caused by potentially toxigenic corynebacterial species.

Surto de abscesso mandibular por Pseudomonas aeruginosa em ovinos / Outbreak of mandibular abscess by Pseudomonas aeruginosa in sheep

Descreve-se um surto de abscesso mandibular em ovelhas da raça Bergamácia no município de Botucatu, estado de São Paulo. Do rebanho de 120 animais, 35 apresentaram aumento de volume mandibular com a presença de nódulos únicos, de consistência pétrea, de diferentes tamanhos, fistulados ou não e sem indicativos de inflamação dos tecidos moles adjacentes. Os animais eram criados em pasto de Panicum maximum cv. Tanzânia com água e sal mineral ad libitum e everminados, via oral, com pistolas dosificadoras. O material para diagnóstico microbiológico e antibiograma foi coletado de cinco animais acometidos, por punção e aspiração dos nódulos. Dos 35 animais acometidos, 19 foram submetidos ao exame radiográfico, um ao exame tomográfico e outro à biópsia óssea da região submandibular. O único ovino que morreu, encontrava-se em estado de caquexia provavelmente devido à localização do aumento de volume que afetou a implantação dos dentes molares daquela região impedindo a apreensão e mastigação adequadas levando a perda da condição corporal e morte. Ao exame necroscópico, observaram-se áreas de necrose caseosa na mandíbula direita de onde isolou-se Pseudomonas aeruginosa. O tratamento utilizado foi baseado na aplicação de iodeto de sódio a 10 por cento por via intramuscular e antibioticoterapia segundo antibiograma com enrofloxacina por via intramuscular, porém com pouca eficácia. Diante do quadro clínico, dos dados de anamnese, da localização das lesões no tecido ósseo mandibular, do resultado do cultivo microbiológico, das alterações radiográficas e tomográficas foi feito o diagnóstico de abscesso mandibular causado por Pseudomonas aeruginosa.

We describe an outbreak of mandibular abscess in Bergamácia sheep from the municipality of Botucatu, São Paulo, Brazil. In a herd of 120 sheep, 35 showed an increase of the mandibular volume with presence of a single nodule of stony consistency and different sizes, often fistulated but with no signs of inflammation in the surrounding soft tissues. The sheep were raised on a Panicum maximum cv. Tanzania pasture, with water and mineral salt ad libitum, and were treated orally against parasites with drenching pistols. Material for microbiological diagnosis and antibiogram was collected from the nodules of five affected sheep, through puncture and aspiration. From 35 affected sheep, 19 were submitted to radiographic examination, one was submitted to computer tomography and another to bone biopsy of the submandibular region. The only sheep that died was in a state of cachexia, probably due to the location of the volume increase which affected the molar hindering apprehension and appropriate chewing. Necropsy revealed areas of caseous necrosis in the jaw, from where Pseudomonas aeruginosa was isolated. The sheep were treated with intramuscular application of 10 percent sodium iodide and the antobiotgic enrofloxacin, but with little efficacy. Based on the clinical data from medical records, the location of lesions in the jaw bone, the result of microbiological culture, radiographic and computer tomography, the diagnosis of mandibular abscesses due to Pseudomonas aeruginosa infection was made.

Paracoccidioidomycosis in a dog: case report of generalized lymphadenomegaly.

Paracoccidioidomycosis (PCM) is a severe systemic mycosis, endemic in Latin America and highly prevalent in Brazil, where it ranks eighth as a mortality cause among infectious and parasitic diseases in humans. The disease in animals has been little explored. It is observed that armadillos can harbor the fungus at high frequencies, although the active disease has not been well documented in this wild mammal. Dogs are susceptible to experimental infection, and the naturally acquired PCM-disease was reported only recently in a dog from Brazil. The present work reports the second case of naturally acquired PCM in a 6-year-old female dog that presented emaciation, lymphadenomegaly, and hepatosplenomegaly. Biochemical and pulmonary radiographic evaluation did not reveal any abnormalities. PCM was diagnosed by clinical findings, culturing, immunohistochemistry, and histopathology of popliteal lymph node. The fungus was recovered from popliteal lymph node, and the molecular analysis showed respective sequencing similarities of 99 and 100% for 803 nucleotides of the Gp43 gene and 592 nucleotides from the ITS-5.8S region of Paracoccidioides brasiliensis. Immunohistochemistry revealed severe lymphadenitis and presented numerous yeasts, which reacted against the gp43 antibody. Histopathology revealed a severe granulomatous lymphadenitis associated with numerous single or multiple budding yeasts. After diagnosis, the dog was successfully treated with itraconazol for 2 years. Veterinarians should be aware of the importance of considering PCM for differential diagnosis, especially in dogs from PCM-endemic areas, whose monophagocytic system involvement is evident.

Microrganismos patogênicos, celularidade e resíduos de antimicrobianos no leite bovino produzido no sistema orgânico: [revisão] / Pathogenic microorganisms, somatic cell count and drug residues evaluation in organic bovine milk: [revision]

Nos últimos anos cresce a preocupação dos consumidores quanto à qualidade do leite e às condições de produção e bem-estar dos animais. Simultaneamente, aumenta o interesse e o consumo de produtos e subprodutosde origem animal produzidos no sistema orgânico, com destaque para o leite e derivados. O presente estudo investigoua presença de microrganismos patogênicos, a sensibilidade e a multi-resistência dos isolados aos antimicrobianos, a celularidade e a presença de resíduos de drogas no leite de vacas, com e sem mastite, produzido no sistema orgânico. Foram amostradas 148 vacas no período médio de lactação, das quais duas com mastite clínica, 72 com mastite subclínica e 74 sem mastite (controles), provenientes de quatro pequenas propriedades do interior do Estado de São Paulo, certificadas como orgânicas. Staphylococcus aureus (25,7%), Streptococcus spp. (21,4%), Corynebacterium bovis (12,9%), Streptococcus agalactiae (4,3%) e Staphylococcus spp. (4,3%) foram os microrganismos mais frequentemente isolados nos animais com mastite. Aspergillus spp. foi isolado de um animal. Ceftiofur (95,2%), oxacilina (84,2%), gentamicina (76,3%) e cefoperazona (70,3%) foram os antimicrobianos mais efetivos frenteaos isolados. As maiores taxas de resistência das linhagens foram constatadas para penicilina (53,5%), ampicilina (41,6%) e neomicina (38,6%). Multi-resistência a trêsou mais fármacos foi encontrada em 40 (39,6%) linhagens. A média da contagem de células somáticas das propriedades para animais com mastite foi 175.742,67 céls/mL, enquantoque para os animais controles foi 58.227,6 céls/mL. A presença de resíduos de antimicrobianos foi observada no leite de quatro (2,7%) animais. Os resultados revelarambaixa celularidade média nos animais, indicativo de boa qualidade do leite. Entretanto, apontam para a necessidadeda adoção de medidas de controle para microrganismos contagiosos e maior...

In last years increase the importance of milk quality and conditions of bovine milking. Simultaneously, increase the interest about organic milk and derivates. The aim of present study was investigate the milk pathogens, sensitivity and multiple drug resistance of isolates, somatic cell count and residues of drugs in milk, from cattle with and without mastitis, come from four little organic dairy farms in State of São Paulo, Brazil. Were used 148 cattle on the middle period of lactation. From these, two showed clinical mastitis, 72 subclinical mastitis and 74 without signs of mammary inflammation (controls). Staphylococcus aureus (25.7%), Streptococcus spp. (21.4%), Corynebacterium bovis (12.9%), Streptococcus agalactiae (4.3%) and Staphylococcus spp. (4.3%) were the more-frequent microorganismsisolated from animals with mastitis. Aspergillus spp. was isolated from one animal. Ceftiofur (95.2%), oxacillin (84.2%), gentamicin (76.3%) and cefoperazone (70.3%) were the moreeffective drugs. High resistance of isolates were found to penicillin (53.5%), ampicillin (41.6%) and neomycin (38.6%). Multiple drug resistance to three or more drugs was observed in 40 (39.6%) isolates. Media of somatic cell count encountered in animals with mastitis and controls were 175,742.67cs/mL and 58,227.6 cs/mL, respectively. Antimicrobials residuesin milk were detected in four (2.7%) animals. The present findings showed the low somatic cell count of animals, indicative of good quality of milk. However, pointed the need of control measures for contagious pathogens of bovine mastitis and more attention forprohibition of antimicrobial use in organic dairy farms.

Prototecose em animais de companhia e aspectos da doença no homem / Protothecosis in companion animals and aspects of the disease in human

Foram revisados dados de etiologia, epidemiologia, patogenia, sinais clínicos, procedimentos diagnósticos, conduta terapêutica e ações de controle e profilaxia da prototecose em cães e gatos, além de aspectos da doença no homem e em bovinos.

Etiology, epidemiology, pathogeny, clinical signs, diagnosis, therapeutic, control and preventive measures in protothecosis in dogs and cats were reviewed, besides aspects of disease in human and bovines.

Nocardiosis: an overview and additional report of 28 cases in cattle and dogs / Nocardiose: visão geral e relato de 28 casos em vacas e cães

Phenotypic characteristics, antimicrobial susceptibility profile, and clinical-epidemiological features of 28 Nocardia strains isolated from 19 cases of bovine mastitis, eight cutaneous-subcutaneous lesions and one case of pneumonia in dogs were evaluated. Microbiological, biochemical, cytological and scanning electron microscopy methods were used in diagnosis. Nocardia asteroides type IV, Nocardia otitidiscaviarum,Nocardia nova (type III) and Nocardia farcinica (type V) were isolated from bovine milk, bronchial lavage and/or cutaneous-subcutaneous abscesses in dogs. Nocardial bovine mastitis was diagnosed predominantly in clinical cases, in dairy herds with poor environmental hygienic conditions between milking and inappropriate intramammary therapy. Canine nocardiosis was observed commonly in animals co-infected with distemper virus. Sulphamethoxazole-trimethoprim (92.8 percent), amikacin (92.8 percent) and ceftiofur (92.8 percent) were the most effective drugs in 28 isolates. Multiple drug resistance to three or more and five or more antimicrobials was observed in ten (35.7 percent) and three (10.7 percent) strains, respectively, predominantly with use of cloxaxillin, cefoperazone and ampicillin. The species (type) classification, clinical-epidemiological characteristics, diagnosis, multiple-drug resistance and public health considerations in Nocardia strains isolated from cattle and dogs in Brazil are discussed, with special reference to report of bovine mastitis by N. otitidiscaviarum by first time in Brazil and the similarity between Nocardia species isolated from human and animal origin.

A caracterização fenotípica, perfil de sensibilidade aos antimicrobianos e aspectos clínico-epidemiológicos foram avaliados em 28 linhagens de Nocardia isoladas de 19 casos de mastite, oito lesões tegumentares e um caso de pneumonia em cão. Foram utilizados no diagnóstico métodos microbiológicos, bioquímicos, citológicos e microscopia eletrônica de varredura. Nocardia asteroides tipo IV, N. otitidiscaviarum,N. nova (tipo III) e N. farcinica (tipo V) foram isoladas do leite de vacas com mastite, de material de lavado transtraqueal e de lesões cutâneas de cães. Nocardiose mamária bovina foi diagnosticada predominantemente sob a forma clínica, em propriedades com precárias condições de higiene na pré e pós-ordenha, e inadequado procedimento de terapia intramamária. Nocardiose canina foi diagnosticada comumente em animais co-infectados com o vírus da cinomose. Sulfametoxazole/trimetoprim (92,8 por cento), amicacina (92,8 por cento) e ceftiofur (92,8 por cento) foram os antimicrobianos mais efetivos frente às linhagens de Nocardia. Resistência múltipla a três ou mais e cinco ou mais antimicrobianos foram observadas, respectivamente, em dez (35,7 por cento) e três (10,7 por cento) linhagens, notadamente frente à cloxacilina, cefoperazona e ampicilina. A caracterização de espécies (tipo), aspectos clínico-epidemiológicos, diagnóstico, resistência múltipla aos antimicrobianos e reflexos em saúde pública de linhagens de Nocardia isoladas de bovinos e cães no Brasil foram discutidos. Foi destacada a similaridade entre as espécies de Nocardia isoladas de animais e do homem, e a primeira descrição no Brasil de N. otitidiscaviarum na etiologia da mastite bovina.

Fatores de risco associados à mastite bovina causada por Prototheca zopfii / Risk factors associated with bovine mastitis caused by Prototheca zopfii

Este trabalho teve como objetivo o estudo de fatores de risco associados à mastite bovina causada por Prototheca zopfii. Foram analisadas 13 propriedades leiteiras dos Estados do Paraná e de São Paulo, segundo os seguintes critérios de seleção: confirmação prévia de casos de mastite por Prototheca spp., triagem pela pesquisa de Prototheca spp. em tanques de expansão e latões e rebanhos com contagem de células somáticas acima de 5x105cel mL-1. As amostras coletadas consistiram de: leite, água, solo, fezes e swab de teteiras. Prototheca spp. foi isolada de amostras de leite dos quartos mamários com mastite clínica ou subclínica em uma propriedade e de amostras de leite e do ambiente em quatro propriedades, nas quais foi isolada em amostras de: água de bebedouro, abastecimento, esgoto, empoçada no piso de estábulo e sala de ordenha, solo de piquete e pasto, teteiras, fezes de bezerros e suínos. Do total de 383 vacas examinadas, Prototheca spp. foi isolada em 20 (5,2 por cento) vacas, sendo caracterizada como P. zopfii em 18. Os fatores de risco associados à mastite causada por P. zopfii foram: criação das vacas a pasto, alimentação dos animais com pasto e silagem, realização de ordenha mecânica em estábulo, permanência das vacas após ordenha em piquete sem alimento, criação de suínos próxima às instalações dos bovinos, existência de cães, gatos e roedores, falta de higienização dos tetos com água, pré-imersão dos tetos em aplicador com retorno e sem a troca do anti-séptico, alimentação dos bezerros com leite de vacas com mastite clínica e serem as vacas da raça holandesa.

This research had as objective the study of risk factors associated with bovine mastitis caused by Prototheca zopfii. Thirteen dairy herds in Paraná and São Paulo states were analyzed and selected according to the following criteria: previous confirmation of Prototheca spp. mastitis cases, screening of Prototheca spp. in bulk tanks and milk cans, and herds with somatic cells count over 5x105cel mL-1. The samples collected consisted of: milk, water, soil, manure and swabs of teat cup rubbers. Prototheca spp. was isolated from mammary quarters with clinical and subclinical mastitis of milk samples in one herd and from the environment and cows in four herds. Out of 383 cows examined, Prototheca spp. was isolated in 20 (5.2 percent) cows with mastitis, and 18 of them were characterized as P. zopfii. In four herds when Prototheca spp. was identified from mammary quarters and environment the agent was isolated from the following samples: water in the waterers, puddled water in the stalls and the milking parlour, supply, sewage, cow pen and pasture soil, teat cup rubbers and manure from calves and swines. The risk factors associated with P. zopfii mastitis consisted of: pasture system, pasture and silage feeding, use of milking machine in stalls, cow pen without fresh feed after milking, raising of swines near bovine housing, existence of dogs, cats and rodents, absence of teats hygienization with water, use of pre-immersion devices with return and without change of antiseptic, calves fed with milk of clinical mastitis cases and the Holstein breed.