O grupo pullorum-gallinarum em provas bacteriologicas comparativas

Foram examinadas 38 amostras de germens do grupo pullorumgallinarum, de origem européa, americana ou isoladas no Brasil, acompanhadas bacteriologicamente durante 3 annos, na fixidez de suas propriedades ou na possibilidade de sua transformação dum typo em outro. a) Distinguiram-se no estudo das propriedades cinco typos de germens no grupo pullorumgallinarum - 1) pullorum gazogeno, 2) pullorum não gazogeno, 3) intermedius, 4) gallinarum ou intermedius? gazogeno, 5) gallinarum não gazogeno. Os 2 primeiros e o 5º já bem cinhecidos e acceitos; o 4º admittido por Beck & Eber, em 1929, o 3º evidenciado por nós em 1935. b) O typo gallinarum gazogeno deve ser mais propriamente talvez considerado como typo intermedius gazogeno a vista de sua acção sobre sorbita e a xylose e sobre o meio de Jordan. c) O quadro final resume as caracteristicas mais importantes destes cinco typos, baseando-se a differenciação principalmente na alteração do vermelho neutro, na produção de H²S, na fermentação de glycerina, rhamnose, xylose, dulcita, sorbita e maltose, na acção sobre o tartaro, na producção de gaz e no aspecto das colonias na superficie de certos meios de cultura. d) Outras propriedades biologicas examinadas - acção sobre o leite, sôro de leite, dextrina, etc, admitidos por varios pesquizadores na differenciação, parecem distituidas de valor. e) As amostras mantiveram fixas as suas propriedades durante todo o tempo em que foram acompanhadas. A hypothese de uma possivel transformação de um typo em outro não foi confirmada em nenhuma das amostras estudadas, justificando a entidade dos varios typos admittidos. f) Discrepancias observadas na fermentação da maltose, referidas por varios pesquizadores com germens desse grupo, não foram confirmadas no presente trabalho. g) Encontrou-se no sôro sanguineo um factor capaz de transformar a maltose em glycose, tornando aquelle assucar fermentescivel. Essa substancia é thermo-estavel e resiste á acção de substancias antidiastasicas. h) A analyse sôrologica das amostras estudadas não permittiu differenciar os typos entre si.

Requirement for cobalamin by Salmonella enterica serovars Typhimurium, Pullorum, Gallinarum and Enteritidis during infection in chickens

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

Molecular differentiation between Salmonella enterica subsp enterica serovar Pullorum and Salmonella enterica subsp enterica serovar Gallinarum

A S. Pullorum (SP) é muito semelhante à S. Gallinarum (SG), agentes da Pulorose e Tifo aviário, respectivamente, sendo que as duas enfermidades são responsáveis por perdas econômicas no setor avícola. SP e SG são de difícil diferenciação em procedimento laboratorial rotineiro, mas uma prova bioquímica muito utilizada na distinção das duas refere-se à capacidade de assimilar o aminoácido ornitina: SP descarboxila este aminoácido enquanto SG não. No entanto, o isolamento de cepas com comportamento bioquímico atípico, tem dificultado tal diferenciação. Um dos genes relacionados à assimilação do aminoácido ornitina, denomina-se gene speC, o qual está presente nos dois sorovares. Analisando 21 amostras de SP e 15 de SG com a utilização da PCR não foi possível realizar a diferenciação dos dois sorovares pois os fragmentos gerados eram idênticos. Posteriormente, com o uso da técnica de tratamento enzimático com a enzima de restrição Eco RI, foi possível observar que o padrão de bandas gerado em cada sorovar era diferente, mesmo quando amostras que apresentavam comportamento bioquímico atípico eram analisadas. Tal fato permitiu a padronização da técnica para ser utilizada na diferenciação entre os sorovares Pullorum e Gallinarum de maneira rápida e segura.

Molecular differentiation of Salmonella Gallinarum and Salmonella Pullorum by RFLP of fliC gene from Brazilian isolates

Although Salmonella Pullorum and Salmonella Gallinarum cause different diseases in poultry, they are very similar. Both are non-motile and present the same somatic antigenic structure. They are differentiated by biochemical tests. Certain atypical strains are very difficult to distinguish. They do not produce the expected results when dulcitol and ornithine descarxboxylase tests are performed. Therefore, additional tests could be helpful. Many studies have chose the part I of the gene that encodes flagellin (fliC) to differentiate serotypes. Most Salmonella strains have two structural genes (fliC and fliB) that encode flagellins. Non-motile strains generally present these structural genes, but are not able to build a functional flagellum. It was demonstrated that enzymatic restriction of the amplified fliC gene using Hinp1I enzyme can differentiate SG from SP. In the present study, this method was adopted to analyze 14 SP and 22 SG strains, including some strains with atypical results in biochemical tests assessing the utilization of dulcitol and ornithine. The results showed that all SG strains were broken by the enzyme, whereas the 14 SP strains were not.

EXAMINATION BY ELISA OF SERA OBTAINED FROM CHICKEN BREEDER AND LAYER FLOCKS SHOWING EVIDENCE OF FOWL TYPHOID OR PULLORUM DISEASE

An indirect ELISA using soluble whole cell antigen was used to screen serum samples obtained from breeder and layer flocks some of which had shown clinical or bacteriological evidence of infection with Salmonella Gallinarum or S. Pullorum. There was good correlation between Salmonella infection and the presence of serum samples showing high optical density (OD) values. Sera from seven flocks showing high values were retested using group D (0-1, 9, 12) lipopolysaccharide (LPS) and g,m-H flagella as detecting antigens. Sera from six flocks produced high OD values with LPS and low values with flagella confirming infection with a non-flagellate, group D Salmonella while one produced high values with both antigens indicating mixed infection with another group D serotype.

SEROLOGICAL RESPONSE OF CHICKENS TO INFECTION WITH SALMONELLA-GALLINARUM S-PULLORUM DETECTED BY ENZYME-LINKED-IMMUNOSORBENT-ASSAY

The serological response to Salmonella pullorum and S. gallinarum infection in chickens was studied with an indirect enzyme-linked immunosorbent assay (ELISA). In broiler chickens, a more virulent strain of S. pullorum produced a significantly lower serum IgG titer than did a less virulent strain. In laying hens, the serum and egg-yolk IgG titers were very similar. In chickens infected with S. gallinarum, high IgG titers persisted for 30 weeks. In chickens reinfected with this strain, each reinfection was followed by transitory increases in IgG lasting no longer than 2 weeks. Serum samples from Brazil taken from a laying flock with evidence of fowl typhoid showed much higher antibody levels than did those from three uninfected flocks. Using lipopolysaccharide as the detecting antigen, infections caused by these salmonellae could be differentiated from those caused by other groups. Incorporation of the appropriate flagella antigen in the ELISA allowed differentiation between infections caused by S. pullorum and S. enteritidis.

Experimental Salmonella enterica serovar Pullorum infection in two commercial varieties of laying hens

An experiment was carried out to investigate the biology of Salmonella Pullorum in two varieties of laying hens, from 5 days of age up to 9 months. One variety was resistant to systemic salmonellosis (light layers producing white eggs) and the other was considered susceptible (brown layers producing brown eggs). The brown birds were more affected by the infection, showing signs of clinical disease in the first month of life. Later, these signs disappeared, but postmortem examination revealed persistent gross pathological changes in the liver, spleen, heart and ovary. The rapid agglutination test detected reactors throughout the experiment, with the strongest agglutination from 1 to 7 months post-infection. S. Pullorum was isolated from some of the organs and the eggs laid throughout the experiment. The relationship between white birds and S. Pullorum was less intense, and there were no noticeable signs of disease. There were few gross pathological changes, and the bacteria were isolated infrequently and only for a brief period after infection, although contaminated eggs were laid by these birds. The strongest serological response in the white chickens occurred between the second and the fifth month post-infection.

Polymerase chain reaction assay based on ratA gene allows differentiation between Salmonella enterica subsp. enterica serovar Gallinarum biovars Gallinarum and Pullorum

Salmonella Pullorum and Salmonella Gallinarum are classified as biovars of Salmonella enterica subsp. enterica serovar Gallinarum. These salmonellae are the causative agents of Pullorum disease and fowl typhoid, respectively, and are widely distributed throughout the world. Although many developed countries have eradicated these diseases from commercial poultry, they are still the cause of significant economic loss in developing countries. When serovar Gallinarum is isolated, it is difficult to immediately differentiate between biovars because they are antigenically identical by serotyping. However, they cause distinct diseases with different epidemiology, and therefore it is important to differentiate them. This may be done biochemically but takes 2 to 3 days. In the present study, S. Pullorum and S. Gallinarum whole genomes were compared, and 1 genomic region of difference, which is part of the ratA gene, was chosen as a molecular marker for a polymerase chain reaction assay to differentiate rapidly between these organisms. In all, 26 strains of S. Gallinarum and 17 S. Pullorum strains were tested and successfully differentiated by the assay. © 2013 The Author(s).

Infecção de aves por mutantes de Salmonella sorotipos gallinarum, pullorum e enteritidis com deleção nos genes cobS E cbiA

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

Análise comparativa dos genomas de Salmonella enterica subsp. enterica sorovar Gallinarum biovares Gallinarum 287/91 e Pullorum 449/87 para identificação de regiões de diferenças (RODs)

Pós-graduação em Microbiologia Agropecuária - FCAV

Diferenciação molecular entre Salmonella enterica subsp enterica sorovar Pullorum e Salmonella enterica subsp enterica sorovar Gallinarum

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Ensaios imunoenzimáticos (ELISA) para detecção da resposta sorológica contra Salmonella Gallinarum, Salmonella Pullorum, Salmonella Enteritidis e Salmonella Typhimurium em aves

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

Identification and characterization of regions of difference between the Salmonella Gallinarum biovar Gallinarum and the Salmonella Gallinarum biovar Pullorum genomes

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

Draft genome sequence of Salmonella enterica subsp. enterica Serovar gallinarum biovar pullorum strain FCAV198, a Brazilian chicken pathogen

Salmonella enterica subsp. enterica serovar Gallinarum biovar Pullorum is a bird-restricted pathogen which causes pullorum disease. The strain FCAV198 was isolated from a pool of chicken ovaries in Brazil, and its genome may be helpful for studies involving molecular mechanisms related to pathogenesis and other related applications.

Indagine sulla presenza di Helicobacter pullorum in allevamenti avicoli italiani

From September 2005 to December 2006, in order to define the prevalence of Helicobacter pullorum in broiler chickens, laying hens and turkey, a total of 365 caecum contents of animals reared in 76 different farms were collected at the slaughterhouse. A caecum content of a ostrich was also sampled. In addition, with the aim of investigating the occurrence of H. pullorum in humans, 151 faeces were collected at the Sant’Orsola-Malpighi University Hospital of Bologna from patients suffering of gastroenteritis. A modified Steele–McDermott membrane filter method was used. Gram-negative curved rod bacteria were preliminary identified as H. pullorum by a PCR assay based on 16S rRNA, then subjected to a RFLP-PCR assay to distinguish between H. pullorum and H. canadensis. One isolate from each farm was randomly selected for phenotypic characterization by biochemical methods and 1D SDSPAGE analysis of whole cell proteins profiles. Minimum Inhibitory Concentration (MIC) for seven different antibiotics were also determined by agar dilution method. Moreover, to examine the intraspecific genomic variability, two strains isolated from 17 different farms were submitted to genotyping by Pulse-Field Gel Electrophoresis (PFGE). In order to assess the molecular basis of fluorquinolone resistance in H. pullorum, gyrA of H. pullorum CIP 104787T was sequenced and nucleotide sequences of the Quinolone Resistance Determining Region (QRDR) of a total of 18 poultry isolates, with different MIC values for ciprofloxacin and nalidixic acid, were compared. According to the PCR and PCR-RFLP results, 306 out of 366 animals examined were positive for H. pullorum (83,6%) and 96,1% of farms resulted infected. All positive samples showed a high number of colonies (>50) phenotipically consistent with H. pullorum on the first isolation media, which suggests that this microrganism, when present, colonizes the poultry caecum at an elevate load. No human sample resulted positive for H. pullorum. The 1D SDS-PAGE whole protein profile analysis showed high similarity among the 74 isolates tested and with the type strain H. pullorum CIP 104787T. Regarding the MIC values, a monomodal distribution was found for ampicillin, chloramphenicol, gentamicin and nalidixic acid, whereas a bimodal trend was noticed for erythromycin, ciprofloxacin and tetracycline (indicating an acquired resistance for these antibiotics). Applying the breakpoints indicated by the CSLI, we may assume that all the H. pullorum tested are sensitive only to gentamicin. The intraspecific genomic variability observed in this study confirm that this species don’t have a clonal population structure, as motioned by other autors. The 2490 bp gyrA gene of H. pullorum CIP104787T with an Open Reading Frame (ORF) encoding a polypeptide of 829 amino acids was for the first time sequenced and characterized. All ciprofloxacin resistant poultry isolates showed ACA®ATA (Thr®Ile) substitution at codon 84 of gyrA corresponding to codons of gyrA 86, 87 and 83 of the Campylobacter jejuni, H. pylori and Escherichia coli, respectively. This substitution was functionally confirmed to be associated with the ciprofloxacin resistant phenotype of poultry isolates. This is the first report of isolation of H. pullorum in turkey and in ostrich, indicating that poultry species are the reservoir of this potential zoonotic microorganisms. In order to understand the potential role as food-borne human pathogen of H. pullorum, further studies must be carried on.