Silvio Luis da Silveira Rocha

Evaluation of selective and non-selective enrichment PCR procedures for Salmonella detection

Aims: To compare PCR combined with enrichment media with the standard microbiological techniques (SMT) and to determine the most sensitive method for the detection of Salmonella and the identification of Salm. typhimurium (ST), Salm. enteritidis (SE), Salm. gallinarum (SG) and Salm. pullorum (SP).

Antibiotic resistance in Salmonella Enteritidis isolated from broiler carcasses

Eighty Salmonella Enteritidis strains isolated from broiler carcasses between May 1995 and April 1996 in the State of Rio Grande do Sul, Brazil, were tested for antibiotic susceptibility using the disk diffusion method. Resistance to colistin, novobiocin, erythromycin and tetracycline was observed in 100% of the isolates. The strains showed intermediate resistance at different levels to kanamycin (1.25%), enrofloxacin (3.75%), neomycin (3.75%), fosfomycin (20%), sulphonamides (86.25%) and nitrofurantoin (90%). Resistance to ciprofloxacin, norfloxacin, gentamicin, polymyxin B, sulphametrim and sulphazotrim was not found. Since resistance to antibiotics especially those introduced in the last decades, was detected, it is recommended that their use must be based on the results of resistance tests or minimum inhibitory concentration tests.

Presence of avipoxvirus DNA in avian dermal squamous cell carcinoma

Dermal squamous cell carcinoma (DSCC; avian keratoacanthoma) is a neoplastic skin lesion of broiler chickens of unknown aetiology. In previous studies, the possibility of the involvement of pox viruses in the cause of DSCC was considered. In this work, a sensitive and specific nested polymerase chain reaction (PCR) protocol was developed that could amplify a 419 base pair DNA fragment of fowlpox virus with a detection limit of less than one infectious unit. Fowlpox virus DNA was always detected in skin samples with fowlpox lesions while it was not detected in samples of unrelated diseases such as cowpox, Mareks disease or infectious laryngotracheitis. Some macroscopically normal skin samples from vaccinated and non-vaccinated birds also produced PCR-positive results, corroborating previous studies on the possibility that a latent or chronic form of fowlpox occurs. Fowlpox virus DNA was consistently detected from DSCC skin lesions, and this finding is discussed.

Detection of virulence-associated genes of Pasteurella multocida isolated from cases of fowl cholera by multiplex-PCR

The current systems of breeding poultry, based on high population density, increase the risk of spreading pathogens, especially those causing respiratory diseases and those that have more than one host. Fowl Cholera (FC) is one such pathogen, and even though it represents one of several avian diseases that should be considered in the differential diagnosis of notifiable diseases that present with sudden death, the pathogenesis and virulence factors involved in FC are still poorly understood. The objective of this study was to investigate twelve genes related to virulence in 25 samples of Pasteurella multocida isolated from FC cases in the southern region of Brazil through the development of multiplex PCR protocols. The protocols developed were capable of detecting all of the proposed genes. The ompH, oma87, sodC, hgbA, hgbB, exBD-tonB and nanB genes were present in 100% of the samples (25/25), the sodA and nanH genes were present in 96% (24/25), ptfA was present in 92% (23/25), and pfhA was present in 60% (15/25). Gene toxA was not identified in any of the samples studied (0/25). Five different genetic profiles were obtained, of which P1 (negative to toxA) was the most common. We concluded that the multiplex-PCR protocols could be useful tools for rapid and simultaneous detection of virulence genes. Despite the high frequency of the analyzed genes and the fact that all samples belonged to the same subspecies of P. multocida, five genetic profiles were observed, which should be confirmed in a study with a larger number of samples.

Genes associated with pathogenicity of avian Escherichia coli (APEC) isolated from respiratory cases of poultry

ABSTRACT .- Rocha A.C.G.P., Rocha S.L.S., Lima-Rosa C.A.V., Souza G.F., MoraesH.L.S., Salle F.O., Moraes L.B. & Salle C.T.P. 2008. Genes associated withpathogenicity of avian Escherichia coli (APEC) isolated from respiratory cases ofpoultry. Pesquisa Veterinaria Brasileira 28(3):183-186. Centro de Diagnostico e Pesquisaem Patologia Aviaria, Departamento de Medicina Animal, Faculdade de Veterinaria,Universidade Federal do Rio Grande do Sul, Av. Bento Goncalves 8824, Porto Alegre,RS 91540-000, Brazil. E-mail: ana.crocha@terra.com.brThe virulence mechanisms of avian pathogenic Escherichia coli (APEC) have beencontinually studied and are believed to be multi-factorial. Certain properties are primarilyassociated with virulent samples and have been identified in avian isolates. In this studya total of 61 E. coli , isolates from chicken flocks with respiratory symptomatology, wereprobed by Polimerase Chain Reation (PCR) for the presence of genes responsible forthe adhesion capacity, P fimbria (

Detecção de Ornithobacterium rhinotracheale (ORT) por meio da reação em cadeia da polimerase (PCR)

Ornithobacterium rhinotracheale (ORT) is a recently discovered Gram negative bacterium that has been associated with respiratory diseases in commercial poultry and wild birds from many countries. In Brazil, antibodies were detected in some broiler and breeder flocks from the States of Sao Paulo and Minas Gerais. Because the bacteria is difficult to grow, the Polymerase Chain Reaction (PCR) has been found to be suitable for identification and diagnostic purposes. The aim of the present work was to verify the occurrence of ORT in Rio Grande do Sul through the detection of the bacteria DNA. Tracheal swabs (84) were collected from 14 broiler flocks of distinct companies. DNA was purified and PCR performed with species specific primers from the ORT 16S ribosomal RNA gene. Amplification products with 784 base pairs were obtained from 10 out of the 84 samples. The positive samples were from four flocks of tree companies established in different regions of the state. The results indicate that this respiratory pathogen occurs in major broiler producing areas from the State of Rio Grande do Sul. Further studies are under way to determine the prevalence of this pathogen and to characterize the strains isolated.

Utilization of immunomagnetic separation for detection of Salmonella in raw broiler parts

This study was conducted aiming to compare the conventional microbiological method to detect Salmonella in broiler parts with the Immunomagnetic Separation method (IMS) followed by plate isolation and also the IMS associated with Rappaport-Vassiliadis broth (RV). The IMS was performed following a pre- enrichment step in buffered peptone water. Sixty-one samples (raw broiler parts) were tested and the results showed that the use of the IMS method alone allowed the isolation of Salmonella in 9 of the tested samples, while the association IMS/ RV detected the agent in 30 samples. The conventional microbiological method was able to isolate the agent in 25 opportunities. These results allowed to conclude that the IMS/RV association presented an increased sensitivity and permitted a better isolation of Salmonella. The conclusion was that other means of isolation, in particular those which do not interfere with the growth of bead bounded Salmonella, should be searched.

Virulence genes and antimicrobial resistance of Pasteurella multocida isolated from poultry and swine

Pasteurella multocida causes atrophic rhinitis in swine and fowl cholera in birds, and is a secondary agent in respiratory syndromes. Pathogenesis and virulence factors involved are still poorly understood. The aim of this study was to detect 22 virulence-associated genes by PCR, including capsular serogroups A, B and D genes and to evaluate the antimicrobial susceptibility of P. multocida strains from poultry and swine. ompH, oma87, plpB, psl, exbD-tonB, fur, hgbA, nanB, sodA, sodC, ptfA were detected in more than 90% of the strains of both hosts. 91% and 92% of avian and swine strains, respectively, were classified in serogroup A. toxA and hsf-1 showed a significant association to serogroup D; pmHAS and pfhA to serogroup A. Gentamicin and amoxicillin were the most effective drugs with susceptibility higher than 97%; however, 76.79% of poultry strains and 85% of swine strains were resistant to sulphonamides. Furthermore, 19.64% and 36.58% of avian and swine strains, respectively, were multi-resistant. Virulence genes studied were not specific to a host and may be the result of horizontal transmission throughout evolution. High multidrug resistance demonstrates the need for responsible use of antimicrobials in animals intended for human consumption, in addition to antimicrobial susceptibility testing to P. multocida.

In vitro efficiency of disinfectants against salmonella enteritidis samples isolated from broiler carcasses

The threat to public health represented by Salmonella is at least partially a consequence of its ecology in poultry hosts. Good manufacturing practices in the processing plant can reduce the contamination of poultry products, and critical control point principles are essential throughout the chain production. One procedure adopted in critical points control to prevent and to reduce Salmonella in farms and poultry products is the use of disinfectants. This study aimed at evaluating disinfectant efficiency against Salmonella enteritidis samples isolated from broiler carcasses in Rio Grande do Sul State between 1995 and 1996. The tested disinfectants were: phenol 1:256, quaternary ammonium 1:2500, glutaraldehyde 1:200, and iodine 1:500, with contact times of 5, 10, 15, and 20 in an in vitro test. .Phenolic compounds showed better results, iodine and glutaraldehyde showed intermediary results, and quaternary ammonium presented efficiency at all contact times evaluated in the in vitro test.

Classification of avian pathogenic Escherichia coli by a novel pathogenicity index based on an animal model

Background: Avian Pathogenic Escherichia coli is the main agent of colibacillosis, a systemic disease that causes considerable economic losses to the poultry industry. In vivo experiments are used to measure the ability of E. coli to be pathogenic. Generally, these experiments have proposed different criteria for results interpretation and did not take into account the death time. The aim of this study was to propose a new methodology for the classification of E. coli pathogenicity by the establishment of a pathogenicity index based in the lethality, death time and the ability of the strain to cause colibacillosis lesions in challenged animals. Materials, Methods & Results: A total of 293 isolates of E. coli were randomly selected to this study. The strains were isolated from cellulitis lesions, broiler bedding material or respiratory diseases and were previously confirmed through biochemical profile. The bacterial isolates were kept frozen at -20°C. The strains were retrieved from stocks and cultured in brain-heart infusion broth overnight at 37°C to obtain a final concentration of 109 UFC/mL. A total of 2940 one-dayold chicks from commercial breeding hens were randomly assigned to groups containing 10 animals and each group was subcutaneously inoculated in the abdominal region with 0.1 mL of the standard inoculum solution containing each of the strains. A control group of 10 broilers were inoculated with 0.1 mL of brain-heart infusion broth by the same route. The chicks were kept for seven days. They were observed at intervals of 6, 12 and 24 h post-inoculation during the first days. From the second day on, the chicks were observed at intervals of 12 h. According to the death time and to the scores of each lesion (aerosaculitis, pericarditis, perihepatitis, peritonitis and cellulitis), a formula to determine the Individual Pathogenicity Index was established. A value of 10 was established as the maximum pathogenicity rate for an inoculated bird. From this rate, 5 points corresponded to scores for gross lesions present at necropsy. For each lesion present, it represents 1 point. The remaining 5 points corresponded to the death time. To obtain the death time value, an index of 1, corresponding to the maximum value assigned to a death on the first day, was divided by the number of days that the birds were evaluated, resulting in a value of 0.1428, which corresponded to a survival bonus factor. It was possible to classify E. coli strains into four pathogenicity groups according to the pathogenicity index: high pathogenicity (pathogenicity index ranging from 7 to 10), intermediate pathogenicity (pathogenicity index ranging from 4 to 6.99), low pathogenicity (pathogenicity index ranging from 1 to 3.99) and apathogenic (pathogenicity index ranging from 0 to 0.99). The analysis of the strains according to their origin revealed that isolates from broiler bedding material presented a lower pathogenicity index. Discussion: It is possible that the source of isolation implies in different results, depending on the criteria adopted. This data reinforces the importance of use a more accurate mathematical model to represents the biological phenomenon. In the study, all avian pathogenic Escherichia coli strains were classified based on a pathogenicity index and the concept of the death time represents an interesting parameter to measure the ability of the strain to promote acute and septicemic manifestation. The use of a support method for poultry veterinary diagnostic accompanying the fluctuation of the bacteria pathogenicity inside the farms may indicate a rational use of antimicrobial in poultry industry.