Caracterização de sorotipos em linhagens do gênero Salmonella isoladas de diferentes afecções em animais domésticos

Foram caracterizados os sorotipos, o perfil de sensibilidade microbiana e os achados clínico-epidemiológicos em 53 linhagens do gênero Salmonella isoladas de 41 cães, nove equinos e três bovinos, acometidos por diferentes manifestações clínicas entre 1997 e 2007. Salmonella Typhimurium (45,3%), Salmonella enterica (22,6%), Salmonella Enteritidis (7,5%), Salmonella enterica subsp enterica 4,5,12i (5,7%), Salmonella Newport (5,7%), Salmonella Dublin (3,8%), Salmonella Agona (3,8%), Salmonella Glostrup (3,8%), Salmonella Saintpaul (1,8%) foram os sorotipos encontrados. Ciprofloxacina (100,0%), norfloxacina (100,0%) e gentamicina (100,0%) foram os antimicrobianos mais efetivos, enquanto a maior resistência das linhagens foi observada para ceftiofur (28,5%) e florfenicol (7,0%). As linhagens foram isoladas de animais com enterite, infecção do trato urinário, septicemia, piometra, pneumonia e conjuntivite. Ressalta-se para o predomínio do sorovar Typhimurium nas diferentes manifestações da salmonelose nos animais. Destaca-se, também, a identificação de sorotipos nos animais que também são observados em casos de salmonelose em humanos

Salmonelose associada à esquistossomose mansônica hépato-esplênica: ação do praziquantel

Cinco pacientes portadores de esquistossomose mansônica hépato-esplênica, associada à salmonelose, foram tratados com dose única de praziquantel (60 mg/kg peso), havendo desaparecimento da hipertermia do 1.° ao 3.° dia após a terapêutica e cura clínica subseqüente da salmonelose e da esquistossomose. O estudo da sensibilidade "in vitro" das bactérias isoladas: Salmonella minnesota, Salmonella dublin, Salmonella panama e Salmonella typhi (2 pacientes) não mostrou ação direta do praziquantel sobre tais enterobactérias. Os soros coletados antes e 24 horas após o tratamento não foram capazes de inibir o crescimento das bactérias isoladas dos respectivos pacientes.

Contribution to the study of diarrhea etiology in neonate dairy calves in São Paulo state, Brazil

Amostras fecais de 203 bezerros com diarréia, idade inferior a 30 dias, de ambos os sexos e de diferentes propriedades do Estado de São Paulo foram examinadas num período de dois anos. Cultivos para pesquisa bacteriana foram feitos em agar acrescido de 10% de sangue bovino e agar Levine. As placas foram incubadas por até 96 horas, em condições aeróbias, a 37°C, com observação dos aspectos de colônia e estudos morfológicos, bioquímicos e realização de outros testes, quando pertinentes. O teste de ELISA foi aplicado para pesquisa de Rotavirus. Cryptosporidium spp. também foi pesquisado e identificado. Resultados revelaram envolvimento de vários patógenos de forma isolada, assim como associados. Rotavirus foi encontrado em 51 (25,1%) das amostras, sendo em 58,8% só, em 41.7% associado a outros microrganismos. Cryptosporydium spp foi isolado em 43 (21.3%) das amostras, sendo só em 65,1% delas e associado a outros enteropatógenos em 34,9%. No exame parasitológico foram encontrados ovos de estrongilídeos em 5 (2,5%) das amostras, não excedendo mais de dois ovos por campo examinado. Ao exame microbiológico, um ou mais microrganismos foram isolados. Escherichia coli foi encontrada em 100% das amostras. As pesquisas de toxina termoestável e do antígeno de aderência K99 realizada nas 73 amostras de E.coli foram negativas, e o grupo sorológico das mesmas foi determinado, sendo 34,2%, 17,8% e 47,9% das amostras pertencentes aos sorogrupos O8, O11 e O101, respectivamente. Salmonella Dublin e Salmonella typhimurium foram isoladas em 5,4% e 6,1% das amostras examinadas, respectivamente.

Efficacy of florfenicol and intravenous fluid therapy for treatment of experimental salmonellosis in newborn calves

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Surto de salmonelose pelo sorovar Dublin em bezerros no Maranhão

Um surto de salmonelose em bezerros causado pela Salmonella enterica subsp. enterica sorovar Dublin é relatado em uma fazenda no município de Timon, Maranhão. De um total de 62 bezerros, 22 (35,5%) adoeceram e destes nove (40,9%) morreram. Os sinais clínicos incluíram febre, depressão, anorexia e, em alguns casos, sinais respiratórios, neurológicos, entéricos ou artrites, com curso clínico hiperagudo ou subagudo. As principais lesões macroscópicas foram hepatomegalia com áreas pálidas multifocais a coalescentes, esplenomegalia e líquido nas cavidades torácica e abdominal. Histologicamente foram observados granulomas paratifoides no fígado, rim e baço, além de trombos e agregados bacterianos em vasos sanguíneos de diversos órgãos. O surto foi controlado com a adoção de antibioticoterapia adequada aliada a correção de algumas medidas sanitárias na propriedade.

Distribution, gene sequence and expression in vivo of the plasmid encoded fimbrial antigen of Salmonella serotype Enteritidis

The pefA gene which encoded the serotype associated plasmid (SAP) mediated fimbrial major subunit antigen of Salmonella enterica serotype Typhimurium shared genetic identity with 128 of 706 salmonella isolates as demonstrated by dot (colony) hybridization. Seventy-seven of 113 isolates of Typhimurium and individual isolates of serotypes Bovis-morbificans, Cholerae-suis and Enteritidis phage type 9b hybridized pefA strongly, whereas 48 isolates of Enteritidis hybridized pefA weakly and one Enteritidis isolate of phage type 14b failed to hybridize. Individual isolates of 294 serotypes and 247 individual isolates of serotype Dublin did not hybridize pefA. Southern hybridization of plasmids extracted from Enteritidis demonstrated that the pefA gene probe hybridized strongly an atypical SAP of 80 kb in size harboured by one Enteritidis isolate of phage-type 9b, whereas the typical SAP of 58 kb in size harboured by 48 Enteritidis isolates hybridized weakly. One Enteritidis isolate of phage type 14b which failed to hybridize pefA in dot (colony) hybridization experiments was demonstrated to be plasmid free. A cosmid library of Enteritidis phage type 4 expressed in Escherichia coli K12 was screened by hybridization for the presence of pef sequences. Recombinant clones which were deduced to harbour the entire pef operon elaborated a PEF-like fimbrial structure at the cell surface. The PEF-like fimbrial antigen was purified from one cosmid clone and used in western blot experiments with sera from chickens infected with Enteritidis phage-type 4. Seroconversion to the fimbrial antigen was observed which indicated that the Enteritidis PEF-like fimbrial structure was expressed at some stage during infection. Nucleotide sequence analysis demonstrated that the pefA alleles of Typhimurium and Enteritidis phage-type 4 shared 76% DNA nucleotide and 82% deduced amino acid sequence identity.

Association between cyclohexane resistance in Salmonella of different serovars and increased, resistance to multiple antibiotics, disinfectants and dyes

A panel of 388 salmonellas of animal and human origin, comprising 35 serotypes, was tested for resistance to cyclohexane and to a range of antibiotics, disinfectants and dyes. Cyclohexane resistance was detected in 41 isolates (10.6%): these comprised members of the serovars Binza (1 of 15), Dublin (1 of 24), Enteritidis (1 of 61), Fischerkietz (4 of 5), Livingstone (9 of 11), Montevideo (1 of 32), Newport (4 of 23), Saint-paul (1 of 3), Senftenberg (10 of 24) and Typhimurium (9 of 93). Most (39 of 41) of the cyclohexane-resistant isolates were from poultry. Statistical analysis showed that the cyclohexane-resistant strains were significantly more resistant than the cyclohexane-susceptible strains to ampicillin, chloramphenicol, ciprofloxacin, erythromycin, nalidixic acid, tetracycline, trimethoprim, cetrimide and triclosan. The multiresistance patterns seen,were typical of those caused by efflux pumps, such as AcrAB. The emergence of such resistance may play an important role in the overall antibiotic resistance picture of Salmonella, with particular effect on ciprofloxacin.

Antibiotic resistance genes, integrons and multiple antibiotic resistance in thirty-five serotypes of Salmonella enterica isolated from humans and animals in the UK

Objectives: To examine 397 strains of Salmonella enterica of human and animal origin comprising 35 serotypes for the presence of aadB, aphAI-IAB, aadA1, aadA2, bla(Carb(2)) or pse1, bla(Tem), cat1, cat2, dhfr1, floR, strA, sul1, sul2, tetA(A), tetA(B) and tetA(G) genes, the presence of class 1 integrons and the relationship of resistance genes to integrons and antibiotic resistance. Results: Some strains were resistant to ampicillin (91), chloramphenicol (85), gentamicin (2), kanamycin (14), spectinomycin (81), streptomycin (119), sulfadiazine (127), tetracycline (108) and trimethoprim (45); 219 strains were susceptible to all antibiotics. bla(Carb(2)), floR and tetA(G) genes were found in S. Typhimurium isolates and one strain of S. Emek only. Class 1 integrons were found in S. Emek, Haifa, Heidelberg, Mbandaka, Newport, Ohio, Stanley, Virchow and in Typhimurium, mainly phage types DT104 and U302. These strains were generally multi-resistant to up to seven antibiotics. Resistance to between three and six antibiotics was also associated with class 1 integron-negative strains of S. Binza, Dublin, Enteritidis, Hadar, Manhattan, Mbandaka, Montevideo, Newport, Typhimurium DT193 and Virchow. Conclusion: The results illustrate specificity of some resistance genes to S. Typhimurium or non- S. Typhimurium serotypes and the involvement of both class 1 integron and non-class 1 integron associated multi-resistance in several serotypes. These data also indicate that the bla(Carb(2)), floR and tetA(G) genes reported in the SG1 region of S. Typhimurium DT104, U302 and some other serotypes are still predominantly limited to S. Typhimurium strains.

Comparative genomics of Salmonella enterica serovars Derby and Mbandaka, two prevalent serovars associated with different livestock species in the UK

Background Despite the frequent isolation of Salmonella enterica sub. enterica serovars Derby and Mbandaka from livestock in the UK and USA little is known about the biological processes maintaining their prevalence. Statistics for Salmonella isolations from livestock production in the UK show that S. Derby is most commonly associated with pigs and turkeys and S. Mbandaka with cattle and chickens. Here we compare the first sequenced genomes of S. Derby and S. Mbandaka as a basis for further analysis of the potential host adaptations that contribute to their distinct host species distributions. Results Comparative functional genomics using the RAST annotation system showed that predominantly mechanisms that relate to metabolite utilisation, in vivo and ex vivo persistence and pathogenesis distinguish S. Derby from S. Mbandaka. Alignment of the genome nucleotide sequences of S. Derby D1 and D2 and S. Mbandaka M1 and M2 with Salmonella pathogenicity islands (SPI) identified unique complements of genes associated with host adaptation. We also describe a new genomic island with a putative role in pathogenesis, SPI-23. SPI-23 is present in several S. enterica serovars, including S. Agona, S. Dublin and S. Gallinarum, it is absent in its entirety from S. Mbandaka. Conclusions We discovered a new 37 Kb genomic island, SPI-23, in the chromosome sequence of S. Derby, encoding 42 ORFS, ten of which are putative TTSS effector proteins. We infer from full-genome synonymous SNP analysis that these two serovars diverged, between 182kya and 625kya coinciding with the divergence of domestic pigs. The differences between the genomes of these serovars suggest they have been exposed to different stresses including, phage, transposons and prolonged externalisation. The two serovars possess distinct complements of metabolic genes; many of which cluster into pathways for catabolism of carbon sources.

CD8(+) T cell adjuvant effects of Salmonella FliCd flagellin in live vaccine vectors or as purified protein

Salmonella flagellin, the flagellum structural subunit, has received particular interest as a vaccine adjuvant conferring enhanced immunogenity to soluble proteins or peptides, both for activation of antibody and cellular immune responses. In the present study, we evaluated the Salmonella enterica FliCd flagellin as a T cell vaccine adjuvant using as model the 9-mer (SYVPSAEQI) synthetic H2(d)-restricted CD8(+) T cell-specific epitope (CS(280-288)) derived from the Plasmodium yoelii circumsporozoite (G) protein. The FliCd adjuvant effects were determined under two different conditions: (i) as recombinant flagella, expressed by orally delivered live S. Dublin vaccine strains expressing the target CS(280-288) peptide fused at the central hypervariable domain, and (ii) as purified protein in acellular vaccines in which flagellin was administered to mice either as a recombinant protein fused or admixed with the target CS(280-288) peptide. The results showed that CS(280-288)-specific cytotoxic CD8(+) T cells were primed when BALB/c mice were orally inoculated with the expressing the CS280-288 epitope S. Dublin vaccine strain. In contrast, mice immunized with purified FliCd admixed with the CS280-288 peptide and, to a lesser extent, fused with the target peptide developed specific cytotoxic CD8(+) T cell responses without the need of a heterologous booster immunization. The CD8(+) T cell adjuvant effects of flagellin, either fused or not with the target peptide, correlated with the in vivo activation of CD11c(+) dendritic cells. Taken together, the present results demonstrate that Salmonella flagellins are flexible adjuvant and induce adaptative immune responses when administered by different routes or vaccine formulations. (C) 2009 Elsevier Ltd. All rights reserved.

Avaliação in vitro da atividade inibitória de Lactobacillus spp., isolados do inglúvio e cecos de aves sobre Salmonella

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Identificação e avaliação in vitro da atividade inibitória de Lactobacillus spp isolados do ingúvio e cecos de aves (Gallus gallus, Linneaus, 1758), em relação a sorotipos de Salmonella

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Virulence of broad- and narrow-host-range Salmonella enterica serovars in the streptomycin-pretreated mouse model.

Salmonella enterica subspecies I serovars are common bacterial pathogens causing diseases ranging from enterocolitis to systemic infections. Some serovars are adapted to specific hosts, whereas others have a broad host range. The molecular mechanisms defining the virulence characteristics and the host range of a given S. enterica serovar are unknown. Streptomycin pretreated mice provide a surrogate host model for studying molecular aspects of the intestinal inflammation (colitis) caused by serovar Typhimurium (S. Hapfelmeier and W. D. Hardt, Trends Microbiol. 13:497-503, 2005). Here, we studied whether this animal model is also useful for studying other S. enterica subspecies I serovars. All three tested strains of the broad-host-range serovar Enteritidis (125109, 5496/98, and 832/99) caused pronounced colitis and systemic infection in streptomycin pretreated mice. Different levels of virulence were observed among three tested strains of the host-adapted serovar Dublin (SARB13, SD2229, and SD3246). Several strains of host restricted serovars were also studied. Two serovar Pullorum strains (X3543 and 449/87) caused intermediate levels of colitis. No intestinal inflammation was observed upon infection with three different serovar Paratyphi A strains (SARB42, 2804/96, and 5314/98) and one serovar Gallinarum strain (X3796). A second serovar Gallinarum strain (287/91) was highly virulent and caused severe colitis. This strain awaits future analysis. In conclusion, the streptomycin pretreated mouse model can provide an additional tool to study virulence factors (i.e., those involved in enteropathogenesis) of various S. enterica subspecies I serovars. Five of these strains (125109, 2229, 287/91, 449/87, and SARB42) are subject of Salmonella genome sequencing projects. The streptomycin pretreated mouse model may be useful for testing hypotheses derived from this genomic data.

Salmonella in companion animals

In an increasingly hygiene concerned society, a major barrier to pet ownership is the perceived role of companion animals in contributing to the risk of exposure to zoonotic bacterial pathogens, such as Salmonella. Manifestations of Salmonella can range from acute gastroenteritis to perfuse enteric fever, in both humans and dogs. Dogs are heavily associated with asymptomatic carriage of Salmonella as the microorganism can persist in the lower intestines of this host which can be then excreted into the environment. Studies in to the asymptomatic carriage of Salmonella in dogs are somewhat dated and there is limited UK data. The current UK carriage rate in dogs was investigated in a randomised dog population and it was revealed that the carriage rate in this population was very low with only one household dog positive for the carriage of Salmonella enterica arizonae (0.2%), out of 490 dogs sampled. Salmonella serotypes share phenotypic and genotypic similarities which are captured in epidemiological typing methods. Therefore, in parallel to the epidemiological investigations, a panel of clinical canine (VLA, UK) and human (Aston University, UK) Salmonella isolates were profiled based on their phenotypic and genotypic characteristics; using API 20E, Biolog Microbial ID System, antibiotic sensitivity testing and PFGE, respectively. Antibiotic sensitivity testing revealed a significant difference between the canine and human isolates with the canine group demonstrating a higher resistance to the panel of antibiotics tested. Further metabolic capabilities of the strains were tested using the Biolog Microbial ID System, which reveal no clear association between the two host groups. However, coupled with Principle Component Analysis two canine isolates were discriminated from the entire population on the basis of a high up-regulation of two carbohydrates. API 20E testing revealed no association between the two host groups. A PFGE harmonised protocol was used to genotypically profile the strains. A dendrogram depicting PFGE profiles of the panel of Salmonella isolates was performed where similarities were calculated by Dice coefficient and represented by UPGMA clustering. Clustering of the profiles from canine isolates and human isolates (HPA, UK) was diverse representing a natural heterogeneity of the genus, additionally, no clear clustering of the isolates was observed between host groups. Clustering was observed with isolates from the same serotype, independent of host origin. Host adaption is a common phenomenon in certain Salmonella serotypes, for example S. Typhi in humans and S. Dublin in cattle. It was of interest to investigate potential host adaptive or restricted strains for canine host by performing adhesion and invasion assays on Dog Intestinal Epithelial Cells (DIECs) (WALTHAM®, UK) and human CaCo-2 (HPA, UK) cell lines. Salmonella arizonae and Enteritidis from an asymptomatic dog and clinical isolate, respectively, demonstrated a significantly high proportion of invasion in DIEC in comparison to human CaCo-2 cells and other tested Salmonella serotypes. This may be suggestive of a potential host restrictive strain as their ability to invade the CaCo-2 cell line was significantly lower than the other serotypes. In conclusion to this thesis the investigations carried out suggest that asymptomatic carriage of Salmonella in UK dogs is low however the microorganism remains as a zoonotic and anthroponotic pathogen based on phenotypic and genotypic characterisation however there may be potential for particular serotype to become host restricted as observed in invasion assays

Isolamento de Salmonella enterica em gambás (Didelphis aurita e Didelphis albiventris) do Estado de São Paulo, Brasil

No Brasil, não há relato de estudos de Salmonella em gambás, sendo assim, este trabalho tem por objetivo determinar a frequência de isolamento de Salmonella enterica em gambás (D. aurita e D. albiventris) no Estado de São Paulo. No período de janeiro de 2005 a dezembro de 2006, foram necropsiados 106 D. aurita e 40 D. albiventris e colhidos fragmentos de intestinos delgado, grosso e suabe da cloaca. As amostras foram plaqueadas diretamente em ágar Mac Conkey, paralelamente suspendidas nos caldos Rappaport-Vassiliadis e Tetrationato e posteriormente plaqueados em ágar XLT4. As colônias sugestivas de Salmonella foram confirmadas através de provas bioquímicas e sorotipagem. Encontrou-se Salmonella enterica em 17,0% (18/106) dos D. aurita. Destes, 50% apresentaram positividade no intestino delgado (ID), 88,9% no intestino grosso (IG) e 66,7% na cloaca. Da espécie S. enterica, as subespécies encontradas foram: diarizonae (11,1%) houtenae e enterica (5,5% cada um); enquanto da subespécie S. enterica enterica os sorotipos foram Newport (83,3%), Typhimurium e Cerro (5,5% cada um). Nos D. albiventris, 17,5% (7/40) eram positivos, sendo que se encontraram 42,8% no ID, 85,7% no IG e 71,4% na cloaca. O sorotipo mais prevalente também foi Newport (71,4%), seguido por Typhimurium, Bareilly e Thompson (14,3% cada um). Através dos resultados obtidos neste estudo pode-se comprovar a presença de Salmonella enterica no trato intestinal de gambás no Brasil.