Multi-resistant diarrheagenic Escherichia coli identified by FTIR and machine learning: a feasible strategy to improve the group classification.

The identification of multidrug-resistant strains from E. coli species responsible for diarrhea in calves still faces many laboratory limitations and is necessary for adequately monitoring the microorganism spread and control. Then, there is a need to develop a screening tool for bacterial strain identification in microbiology laboratories, which must show easy implementation, fast response, and accurate results. The use of FTIR spectroscopy to identify microorganisms has been successfully demonstrated in the literature, including many bacterial strains; here, we explored the FTIR potential for multi-resistant E. coli identification. First, we applied principal component analysis to observe the group formation tendency; the first results showed no clustering tendency with a messy sample score distribution; then, we improved these results by adequately selecting the main principal components which most contribute to group separation. Finally, using machine learning algorithms, a predicting model showed 75% overall accuracy, demonstrating the method's viability as a screaming test for microorganism identification.

Phytase and xylanase in diets with nutritional adjustments and their effects on serum biochemistry, morphometry and intestinal health of broilers.

To examine the association between phytase and xylanase in diets with nutritional adjustments on intestinal morphometry, serum biochemistry and microbiology of broilers, 250 broilers were evaluated in a completely randomized design with five treatments and five replicates. The following treatments were tested: positive control diet - without phytase or xylanase; negative control diet - with an energy reduction of 100 kcal/kg, without phytase or xylanase; and three diets containing xylanase and phytase and energy reductions of 50, 100 and 150 Kcal/kg. For all energy-reduced diets, the nutritional matrix of phytase with phosphorus (0.15%), calcium (0.165%) and sodium (0.035%) was considered. An effect of the association between enzyme inclusion and metabolizable energy reduction in the diets was observed only by contrast analysis, for villus height. Intestinal health was not changed. Only the serum phosphorus concentration was altered by the treatments at the different evaluated ages. The association of phytase (500 FTU/kg) with xylanase (16000 BXU/kg) in diets with reductions of up to 150 Kcal/kg metabolizable energy, 0.15% digestible P, 0.165% Ca and 0.035% Na does not alter the intestinal morphometry, serum biochemistry or microbiology of broilers.

Outbreak investigation of septicemic salmonellosis in calves.

INTRODUCTION: An early and accurate diagnosis of septicemic salmonellosis is critical for implementing timely and proper treatment, prevention, and control measures. METHODOLOGY: Here, we report a study on three outbreaks of septicemic salmonellosis in calves from Midwestern Brazil. RESULTS: the morbidity, mortality and lethality rates were of 10.55%, 2.79%, and 26.4%, respectively. Higher susceptibility was detected in Bos taurus than in Bos indicus cattle. Clinical manifestations consisted of apathy, hyperthermia, difficulty breathing and panting, and pallor of the mucous membranes. Chronic cases had necrosis of the tail tip and ears. Gross findings included enlarged liver, non-collapsed edematous lungs and diphtheritic enteritis. Significant histopathological changes included paratyphoid nodules in the liver and acute interstitial pneumonia. Salmonella enterica subsp. enterica serotype Dublin was detected by culture and by PCR from the blood of live calves, and from the spleen, liver, bile, mesenteric lymph node and lung samples of necropsied calves. CONCLUSIONS: We suggest that in clinical cases of septicemic salmonellosis, blood samples are better than fecal samples for detection of the agent, being a sound test to identify animal carriers in the herd.

Salmonellosis in calves without intestinal lesions / Salmonelose em bezerros sem manifestações intestinais

Salmonellosis is a known cause of enteric disorders in calves. However, cases in the septicemic form may not present enteric lesions, which may lead the veterinary practitioner to not suspect salmonellosis, compromising the diagnosis. The current study describes the epidemiological, clinical, pathological and immunohistochemical aspects of septicemic salmonellosis in calves without enteric lesions. The protocols involving bovine material submitted to the Pathology Laboratory (LAP) of the "Faculdade de Medicina Veterinária e Zootecnia" (FAMEZ) of the "Universidade Federal do Mato Grosso do Sul" (UFMS) from January 1995 to July 2018 were studied. Cases confirmed or suggestive of septicemic salmonellosis in calves without enteric manifestations were selected. Fragments of the liver, lung, and spleen embedded in paraffin were submitted to immunohistochemistry (IHC). Only cases in which there was positive marking on the IHC or culture isolation of Salmonella were included in this study. Of a total of 5,550 cattle examined in the period, ten presented septicemic salmonellosis without enteric lesions. Clinical signs included mucosal pallor, apathy, hyperthermia, and dyspnea. Only three calves presented diarrhea, and two were found dead before clinical changes were observed. The most common necropsy findings were hepatosplenomegaly; yellow, orange or brown discolored livers; pale mucous membranes; inflated and sometimes red lungs; fibrin or fluid within body cavities; and gallbladder filled with inspissated bile. Jaundice was observed in three calves that had a concomitant infection with Anaplasma sp. Microscopically, paratyphoid hepatic nodules and interstitial pneumonia were the most frequent manifestations, followed by thrombosis and bacterial colonies in the spleen, lung, liver, and brain. A strong positive marking was observed in IHC, predominantly in the lung and to a lesser extent in the liver. Polymerase chain reaction (PCR) indicated the Dublin serotype as the causative agent in the samples of the four calves submitted to this procedure. In calves, the septicemic form was the major cause of death due to salmonellosis. Septicemic salmonellosis was usually not accompanied by diarrhea. The clinical signs of septicemia are nonspecific and of little assistance in the diagnosis. IHC has been shown to be efficient in the detection of the agent, mainly in the lung and especially in situations where it is not possible to perform bacterial culture.(AU)

A salmonelose é uma causa conhecida de distúrbios entéricos em bezerros. Porém, casos na forma septicêmica podem não apresentar manifestação entérica, o que leva o médico veterinário a não suspeitar de salmonelose, comprometendo o diagnóstico. Este estudo descreve os aspectos epidemiológicos, clínicos, patológicos e imuno-histoquímicos da salmonelose septicêmica em bezerros sem lesões entéricas. O estudo foi realizado a partir dos protocolos referentes a materiais de bovinos enviados para diagnóstico ao Laboratório de Anatomia Patológica (LAP) da Faculdade de Medicina Veterinária e Zootecnia (FAMEZ) da Universidade Federal de Mato Grosso do Sul (UFMS) de janeiro de 1995 a julho de 2018. Foram selecionados os casos de bezerros confirmados ou sugestivos de salmonelose septicêmica sem lesões entéricas. Fragmentos de fígado, pulmão e baço embebidos em parafina foram submetidos ao exame de imuno-histoquímica (IHQ). Somente foram incluídos neste estudo casos em que houve marcação positiva na IHQ ou isolamento da bactéria em cultura. De um total de 5.550 bovinos examinados no período, dez apresentaram salmonelose septicêmica sem lesão entérica. Os sinais clínicos incluíram palidez de mucosas, apatia, hipertermia e dispneia. Apenas três bezerros apresentaram diarreia e dois foram encontrados mortos sem terem sido observadas alterações clínicas. Os achados mais frequentes de necropsia foram hepatoesplenomegalia, fígado amarelado, alaranjado ou acastanhado, palidez de mucosas, pulmões inflados e, por vezes, vermelhos, fibrina ou líquido nas cavidades do organismo e vesícula biliar repleta de bile grumosa. Icterícia foi observada em três bezerros que apresentavam infecção concomitante por Anaplasma sp. Microscopicamente, os nódulos paratifoides hepáticos e pneumonia intersticial foram as manifestações mais encontradas, seguidas por trombose e colônias bacterianas no baço, pulmão, fígado e encéfalo. Na IHQ, marcação fortemente positiva foi observada, predominantemente, no pulmão e, em menor intensidade, no fígado. A técnica de reação em cadeia de polimerase (PCR) tipificou o sorotipo Dublin como agente etiológico nas amostras dos quatro bezerros submetidos a este procedimento. Em bezerros, a forma septicêmica foi a principal responsável pelas mortes por salmonelose. Na maioria das vezes essa forma não estava acompanhada por diarreia. Os sinais clínicos da forma septicêmica são inespecíficos e de pouco auxílio no direcionamento do diagnóstico. A IHQ mostrou-se eficiente na detecção do agente principalmente no pulmão e especialmente nas situações em que não é possível a realização da cultura bacteriana.(AU)

Photoinactivation effect of eosin methylene blue and chlorophyllin sodium-copper against Staphylococcus aureus and Escherichia coli.

The use of eosin methylene blue according to Giemsa as photosensitizer is presented for the first time in this paper. The present study evaluated the potential application of chlorophyllin sodium copper salt (CuChlNa) and eosin methylene blue according to Giemsa (EMB) as antimicrobial photosensitizers (aPS) for photodynamic inactivation (PDI) of Staphylococcus aureus (gram-positive) and Escherichia coli (gram-negative) bacteria. The experiments were performed using S. aureus stain ATCC 25923 and E. coli ATCC 25922 in which five aPS concentrations (0.0, 1.0, 2.5, 5.0, 10.0, and 20.0 µM for S. aureus and 0.0, 5.0, 10.0, 20.0, 40.0, and 50.0 µM for E. coli) were prepared and added in 2 mL of a saline solution containing the bacterial inoculum. After aPS incubation, the samples were divided into two groups, one kept in the dark and another submitted to the illumination. Then, the bacterial inactivation was determined 18 h after the incubation at 37 °C by counting the colony-forming units (CFU). The results revealed that both EMB and CuChlNa can be used as aPS for the photoinactivation of S. aureus, while only EMB was able to photoinactivate E. coli. Nevertheless, a more complex experimental setup was needed for photoinactivation of E. coli. The data showed that EMB and CuChlNa presented similar photoinactivation effects on S. aureus, in which bacterial growth was completely inhibited at photosensitizer (PS) concentrations over 5 µM, when samples were previously incubated for 30 min and irradiated by a light dose of 30 J cm-2 as a result of an illumination of 1 h at 8.3 mW cm-2 by using a red light at 625 nm with a 1 cm beam diameter and output power of 6.5 mW. In the case of E. coli, bacterial growth was completely inhibited only when combining a PS incubation period of 120 min with concentrations over 20 µM.

Atividade antibacteriana in vitro da própolis marrom / In vitro biological activity of brown propolis

Objetivou-se avaliar a atividade antibacteriana in vitro da própolis marrom, por meio da determinação da Concentração Inibitória Mínima (CIM). O extrato alcoólico de própolis foi obtido de 35 g de própolis bruta macerada em 65 mL de álcool de cereais. As concentrações do extrato de própolis usadas foram: 75mg/mL; 56,4mg/mL; 37,5mg/mL; 18,9mg/mL; 9,3mg/mL; 4,5mg/mL e 2,25mg/mL. Foram utilizados 32 isolados de bactérias Gram-positivas: Rhodococcus equi, Staphylococcus aureus, Staphylococcus hyicus, Staphylococcus spp. e Streptococcus spp., e 32 isolados de bactérias Gram-negativas: Enterobacter agglomerans, Escherichia coli, Klebsiella pneumoniae, Klebsiella sp., Pseudomonas aeruginosa, Pseudomonas spp., Salmonella spp. e Serratia rubidaea, provenientes de processos clínicos infecciosos de animais domésticos, obtidas e armazenadas no Laboratório de Bacteriologia da FAMEZ/UFMS. O extrato de própolis marrom apresentou atividade antimicrobiana com CIM variando de 2,25 a 18,9mg/mL para as bactérias Gram-positivas e 4,5 a 18,9mg/mL para as bactérias Gram-negativas, sendo as bactérias provenientes de bovinos e caninos as mais resistentes. Conclui-se que a própolis marrom tem ação bactericida, em função da espécie da bactéria e da procedência animal.

The aim of this study was to evaluate the in vitro antibacterial activity of brown propolis, by determining the minimum inhibitory concentration (MIC). The alcoholic extract of propolis was obtained from 35g of crude propolis macerated in 65mL of cereals alcohol. Dilutions of propolis extracts used were: 75mg/mL; 56.4mg/mL; 37.5mg/mL; 18.9mg/mL; 9.3mg/mL; 4.5mg/mL, and 2.25mg/mL. Thirty-two Gram-positive bacteria isolates were used: Rhodococcus equi, Staphylococcus aureus, Staphylococcus hyicus, Staphylococcus spp. and Streptococcus spp.; and the thirty-two Gram-negative bacteria isolated were: Enterobacter agglomerans, Escherichia coli, Klebsiella pneumoniae, Klebsiella sp., Pseudomonas aeruginosa, Pseudomonas sp., Salmonella sp., and Serratia rubidaea, obtained from infectious clinical processes of domestic animals, and stored in the Laboratory of Bacteriology of FAMEZ/UFMS. The brown propolis extract showed antibacterial activity with MIC ranging from 2.25 to 18.9mg/ml for Gram-positive bacteria and 4.5 to 18.9mg/ml for Gram-negative bacteria, and bacteria from cattle and dogs were more resistant. It was concluded that brown propolis has antibacterial action, but the effect depends on the species of the bacterium and its origin.

Estimativas de perdas econômicas causadas por reação granulomatosa local após uso de vacina oleosa contra febre aftosa em bovinos de Mato Grosso do Sul / Estimates of economic losses caused by local granulomatous reaction after use of an oily vaccine against FMD in cattle of Mato Grosso do Sul

Lesões exacerbadas à vacina contra a febre aftosa foram observadas em 1.815 de um total de 5.000 bovinos abatidos em frigorífico com inspeção federal. Essas lesões resultaram na condenação de parte das carcaças de acordo com o Regulamento da Inspeção Industrial e Sanitária de Produtos de Origem Animal. Os bovinos haviam sido vacinados em maio de 2012 e abatidos em setembro do mesmo ano e divididos em Lotes 1 e 2 constituídos respectivamente por 1.500 e 315 bovinos. As lesões de reação vacinal eram caracterizadas por nódulos protuberantes, circunscritos, bem delimitados, firmes e de tamanhos variáveis, distribuídos multifocalmente na musculatura do aspecto lateral do pescoço; ao corte apresentavam exsudato purulento. Amostras da lesão de 28 bovinos foram avaliadas histologicamente e os principais achados foram piogranulomas com espaços claros circulares de tamanhos variados no centro, circundados por intenso infiltrado de neutrófilos íntegros e necróticos, circundados por macrófagos epitelioides com citoplasma vacuolizado, ocasionais células gigantes multinucleadas e, mais externamente, por abundante tecido conjuntivo em meio observavam-se linfócitos e plasmócitos. Culturas bacterianas realizadas em amostras do exsudato da lesão de seis bovinos resultaram negativas. Quando as carcaças afetadas foram submetidas ao toalete foi retirado em média 1,8 e 2,0 kg de músculo da área afetada, respectivamente dos Lotes 1 e 2. O prejuízo econômico da propriedade de origem dos bovinos afetados foi de R$ 20.424,00, considerando o preço pago pela arroba do boi no mês e ano da ocorrência. Esses valores à época seriam suficientes para adquirir 29,17 bezerros desmamados para engorda. Se considerarmos 5.000 bovinos vacinados forem considerados como população sob risco o coeficiente de morbidade seria de 0,36%. Os resultados deste estudo demonstram que perdas por reação vacinal, mesmo quando não provocam sinais clínicos marcantes, podem ocasionar importantes prejuízos econômicos...

Lesions caused by excessive reaction to foot and mouth vaccine were observed in 1,815 out of 5,000 cattle slaughtered in a slaughterhouse under federal meat inspection. Such lesions resulted in condemnation of the affect parts of the carcasses in accordance with the Brazilian Federal Meat Inspection Regulations. The affected cattle had been vaccinated on May 2012 and then slaughtered in September of the same year. They were allotted to two groups with 1,500 (Groups 1) and 315 cattle (Group 2). Post vaccination lesions were characterized as salient, firm variable sized nodules multifocally distributed in the musculature of the lateral neck. Pus oozed at the cut surface of these nodules. Samples of 28 cattle were submitted for histopathological examination; main findings were pyogranulomas with central variable-sized clear spaces surrounded by marked infiltrate of viable and dead neutrophils, which were surrounded by epithelioid macrophages with vacuolated cytoplasm and occasional multinucleated giant cell. This inflammatory reaction was walled by abundant connective tissue infiltrated by lymphocytes and plasma cells. Samples of the lesion of six cattle were submitted to bacteriological culture with negative results. When affected carcasses went through dressing, there was an average of 1.8 and 2.0 kg of muscle tissue cut off from the carcasses respectively of Group 1 and 2. Economic losses in the farm of origin of the affected cattle was R$ 20,424.00, considering the price paid by kg of cattle for slaughter at the time of the occurrence. This amount at that time was enough to buy 29.17 weaned calves for fattening. If one considers theses 5,000 cattle vaccinated on the farm as the population at risk, the morbidity rate would be 0.36%. These results indicate that the losses due to vaccine reaction, even with no overt clinical signs, may cause important economic losses...

Doenças de ovinos diagnosticadas no Laboratório de Anatomia Patológica Animal da Universidade Federal de Mato Grosso do Sul (1996-2010) / Sheep diseases diagnosed at the Laboratory of Animal Pathology, Federal University of Mato Grosso do Sul, Brazil (1996-2010)

Nas últimas décadas a pecuária ovina cresceu significativamente no Brasil. Concomitantemente, grupos de pesquisas e laboratórios de diagnósticos realizam estudos retrospectivos com a finalidade de fornecer subsídios técnico-científicos para os médicos veterinários. Desta forma, realizou-se um estudo de prevalência nos arquivos do Laboratório de Anatomia Patológica Animal (LAP) da Universidade Federal de Mato Grosso do Sul (UFMS) no período de Janeiro de 1996 a Dezembro de 2010. O Laboratório de Bacteriologia da UFMS e o Setor de Patologia Veterinária da Universidade Federal do Rio Grande do Sul forneceram apoio diagnóstico nos casos de mannheimiose pulmonar e scrapie, respectivamente. Os laudos da espécie ovina foram revisados e agrupados em conclusivos e inconclusivos, dos quais foram excluídos os casos experimentais e de outros estados e países. Os casos conclusivos foram classificados de acordo com a etiologia da doença. Os exames da espécie ovina somaram 331 laudos (3,97 %) de um total de 8.333 casos diagnosticados no período. Destes, foram excluídos sessenta e quatro (19,3%) casos experimentais e materiais oriundos de outros estados ou países. Dos 267 casos remanescentes, 87 (32,6%) foram inconclusivos e 180 (67,4%) considerados conclusivos, sendo 60 (33,3%) doenças infecciosas e parasitárias; 45 (25%) intoxicações e toxi-infecções; 41 (22,8%) "lesões sem causa definida"; 22 (12,2%) doenças metabólicas e nutricionais; 10 (5,6%) foram classificadas como "outros distúrbios" e 2 (1,1%) neoplasmas. A hemoncose, intoxicação por Brachiaria spp., pleuropneumonias, broncopneumonias, pneumonias fibrinonecrosante ou fibrinossupurativa sem causa definida e a intoxicação por cobre foram as doenças mais prevalentes no período estudado. Dois casos de scrapie foram diagnosticados no período.

Sheep farming has increased significantly in Brazil during the last decades. Concurrently, research groups and diagnostic laboratories compile data and perform retrospective studies to provide important insight for professionals. A prevalence study from January 1996 to December 2010 was carried out in the archives of Laboratório de Anatomia Patológica Animal (LAP), Universidade Federal de Mato Grosso do Sul (UFMS). Laboratório de Bacteriologia, UFMS, and Setor de Patologia Veterinária at Universidade Federal do Rio Grande do Sul helped on the diagnostic of pulmonary mannheimiosis and scrapie respectively. The reports for sheep were reviewed and grouped into conclusive and inconclusive ones. The conclusive cases were classified according to the etiology of the disease. In the period, 331 exams (3.97%) were done. Sixty-four experimental cases and materials from other states or countries (19.3%) were excluded. Remaining cases (267), eighty-seven (32.6%) were inconclusive and 180 (67.4%) were considered conclusive reports, were classified according to the etiology: 60 (33.3%) infectious and parasitary diseases; 45 (25%) were poisonings and toxi-infections; 41 (22.8%) were summarized as "injuries without apparent cause"; 22 (12.2%) cases of metabolic and nutritional diseases; 10 (5.6%) were classified as "other disorders" and 2 (1.1%) case of neoplasms. Haemonchosis, fibrinonecrotic or fibrinopurulent pleuropneumonia, bronchopneumonia and pneumonia, poisonings by Brachiaria spp. and copper poisoning were the most prevalent diseases in sheep. Two cases of scrapie have been diagnosed in this period.

Evidence that lipopolisaccharide may contribute to the cytokine storm and cellular activation in patients with visceral leishmaniasis.

BACKGROUND: Visceral leishmaniasis (VL) is characterized by parasite-specific immunosuppression besides an intense pro-inflammatory response. Lipopolisaccharide (LPS) has been implicated in the immune activation of T-cell deficient diseases such as HIV/AIDS and idiopathic lymphocytopenia. The source of LPS is gram-negative bacteria that enter the circulation because of immunological mucosal barrier breakdown. As gut parasitization also occurs in VL, it was hypothesized that LPS may be elevated in leishmaniasis, contributing to cell activation. METHODOLOGY/PRINCIPAL FINDINGS: Flow cytometry analysis and immunoassays (ELISA and luminex micro-beads system) were used to quantify T-cells and soluble factors. Higher LPS and soluble CD14 levels were observed in active VL in comparison to healthy subjects, indicating that LPS was bioactive; there was a positive correlation between these molecules (r = 0.61;p<0.05). Interestingly, LPS was negatively correlated with CD4(+) (r = -0.71;p<0.01) and CD8(+) T-cells (r = -0.65;p<0.05). Moreover, higher levels of activation-associated molecules (HLA-DR, CD38, CD25) were seen on T lymphocytes, which were positively associated with LPS levels. Pro-inflammatory cytokines and macrophage migration inhibitory factor (MIF) were also augmented in VL patients. Consistent with the higher immune activation status, LPS levels were positively correlated with the inflammatory cytokines IL-6 (r = 0.63;p<0.05), IL-8 (r = 0.89;p<0.05), and MIF (r = 0.64;p<0.05). Also, higher plasma intestinal fatty acid binding protein (IFABP) levels were observed in VL patients, which correlated with LPS levels (r = 0.57;p<0.05). CONCLUSIONS/SIGNIFICANCE: Elevated levels of LPS in VL, in correlation with T-cell activation and elevated pro-inflammatory cytokines and MIF indicate that this bacterial product may contribute to the impairment in immune effector function. The cytokine storm and chronic immune hyperactivation status may contribute to the observed T-cell depletion. LPS probably originates from microbial translocation as suggested by IFABP levels and, along with Leishmania antigen-mediated immune suppression, may play a role in the immunopathogenesis of VL. These findings point to possible benefits of antimicrobial prophylaxis in conjunction with anti-Leishmania therapy.

Genetic and antigenic analysis of Babesia bigemina isolates from five geographical regions of Brazil

A molecular epidemiological study was performed with Babesia bigemina isolates from five geographical regions of Brazil. The genetic analysis was done with random amplification of polymorphic DNA (RAPD), repetitive extragenic palindromic elements-polymerase chain reaction (REP-PCR) and enterobacterial repetitive intergenic consensus sequences-polymerase chain reaction (ERIC-PCR) that showed genetic polymorphism between these isolates and generated fingerprinting. In RAPD, ILO872 and ILO876 primers were able to detect at least one fingerprinting for each B. bigemina isolate. The amplification of B. bigemina DNA fragments by REP-PCR and ERIC-PCR gave evidence for the presence in this haemoprotozoan of the sequences described previously in microorganisms of the bacterial kingdom. For the first time it was demonstrated that both techniques can be used for genetic analysis of a protozoan parasite, although the ERIC-PCR was more discriminatory than REP-PCR. The dendogram with similarity coefficient among isolates showed two clusters and one subcluster. The Northeastern and Mid-Western isolates showed the greatest genetic diversity, while the Southeastern and Southern isolates were the closest. The antigenic analysis was done through indirect fluorescent antibody technique and Western blotting using a panel of monoclonal antibodies directed against epitopes on the merozoite membrane surface, rhoptries and membrane of infected erythrocytes. As expected, the merozoite variable surface antigens, major surface antigen (MSA)-1 and MSA-2 showed antigenic diversity. However, B cell epitopes on rhoptries and infected erythrocytes were conserved among all isolates studied. In this study it was possible to identify variable and conserved antigens, which had already been described as potential immunogens. Considering that an attenuated Babesia clone used as immunogen selected populations capable of evading the immunity induced by this vaccine, it is necessary to evaluate more deeply the cross-protection conferred by genetically more distant Brazilian B. bigemina isolates and make an evaluation of the polymorphism degree of variable antigens such as MSA-1 and MSA-2