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.

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.

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.

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.

RESUMOOrnithobacterium rhinotracheale (ORT) é uma bactéria Gram negativa recentemente descrita que se encontra associada às doenças do trato respiratório em criações de aves comerciais e silvestres em vários países do mundo. No Brasil, foram detectados anticorpos em um pequeno número de frangos de corte e suas matrizes dos Estados de São Paulo e Minas Gerais. Como a bactéria é fastidiosa, a Reação em Cadeia da Polimerase (PCR) torna-se útil para sua detecção e identificação. O presente trabalho visou verificar a ocorrência da ORT no Rio Grande do Sul pela detecção do DNA da bactéria. Foram coletadas 84 amostras de suabe de traquéia de aves pertencentes a 14 lotes de diferentes empresas avícolas. O DNA foi purificado e a PCR realizada com iniciadores específicos para o gene do RNA ribossomal 16S da ORT. Foram observados produtos de amplificação com 784 pares de bases em 10 das 84 amostras. As amostras positivas pertenciam a quatro lotes de três empresas estabelecidas em diferentes regiões do RS. Os resultados indicam que este patógeno respiratório de aves existe no Brasil e está presente em importantes regiões criatórias do RS. Outros estudos estão em andamento para determinar a prevalência e caracterização dos isolados obtidos.Palavras-chave: Ornithobacterium rhinotracheale, patologia aviária, PCR, detecção, identificação. ABSTRACTOrnithobacterium 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 São 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.

Fowl Cholera (FC) is a disease caused by Pasteurella multocida. The severity of this disease is partly caused by virulence factors. Genes encoding fimbriae, capsule, sialidases and proteins for iron metabolism may be related to P. multocida’s ability to infect the host. Besides to examining DNA for the presence of virulence genes, DNA is essential for the diagnostic and FTA cards are an alternative for genetic material transport. The study aims to evaluate the viability of P. multocida DNA transport using the cards and to detect 14 virulence genes in 27 strains isolated from FC cases in the United States by multiplex-PCR. No growth was observed in any of the FTA cards, which was essential to assess the security. Furthermore, DNA detection was possible in 100% of the samples, independent of the storage period (7 to 35 days) and temperature (4°C and 37°C). ptfA, exbd-tonB, hgbA, nanB, oma87, hyaD-hyaC, sodC, hgbB, sodA, nanH and pfhA genes were detected in more than 80% of the samples. FTA cards have proven to be a viable and safe tool for DNA transport of P. multocida. A majority of genes showed a high frequency, which was similar to strains isolated from FC cases.

Utilização de inteligência artificial (redes neurais artificiais) para a classificação do comportamento bioquímico de amostras de Escherichia coli isoladas de frangos de corte* The use of artificial intelligence (artificial neural networks) to classify the biochemical reactions of Escherichia coli isolates from broilers

Background: Avian pathogenic E. coli (APEC) and uropathogenic E. coli (UPEC) are responsible, respectively, for avian colibacillosis and for 80% of urinary tract infections in humans. E. coli control is difficult due to the absence of a reliable method to differentiate pathogenic and commensal strains. Genetic similarity between APEC and UPEC suggests a common ancestral origin and the capability of potentially pathogenic strains to affect human health. The classification in phylogenetic groups facilitates the identification of pathogenic strains. The objective of this work was to classify APEC and UPEC E. coli strains into phylogenetic groups and to associate it with in vivo pathogenicity.Materials, Methods & Results: 460 APEC and 450 UPEC strains, stored in BHI with glycerol at -80°C, were selected. APEC strains were isolated from cellulitis, respiratory tract and poultry litter of broiler flocks from Southern Brazil. The UPEC strains from urinary tract infection were provided by a hospital in Porto Alegre. After DNA extraction, APEC and UPEC strains were classified into four phylogenetic groups (A, B1, B2 and D) by a multiplex-PCR protocol for the detection of the chuA and yjaA genes and the TspE4.C2 DNA fragment. Phylogenetic groups were associated with pathogenicity indexes (PI), presented on a scale of 0 to 10, which were previously obtained through the inoculation of APEC strains in one-day-old chicks. Phylogenetic groups were also associated with the presence of 38 virulence-associated genes. The multiplex-PCR protocol was able to differentiate 100% of the APEC and UPEC strains in the four phylogenetic groups. The majority of APEC strains were classified into phylogenetic groups D (31.1%) and B2 (24.1%). On the other hand, the majority of UPEC strains were classified into B2 (53.6%). Among APEC strains, five genes (crl, mat, ompA, fimC and fimH) were detected in more than 80% of strains in all groups. Some genes showed a significant association with specific phylogenetic groups. Gene ireA was exclusively to group D, kpsMT II and cvaC to B2 and sat was exclusively to B1. Four genes (ireA, sfa/focCD, ibeA, tsh) were detected in more than 70% of UPEC strains in all phylogenetic groups. Gene iroN1 showed a significant association exclusively to group A, and iucD, papC and irp2 to B1 group. APEC isolated from poultry litter presented significantly lower PIs than those isolated from cellulitis and from birds with respiratory signs. The average PI from B2 group was significantly higher than that of D group. In addition, the PIs of the two groups were significantly higher than those of A and B1.Discussion: The high frequency of UPEC classified as B2 is in agreement with the literature. More virulent strains are usually classified into B2 group and some of them may be classified into D group. On the other hand, the distribution of APEC isolates in phylogenetic groups is characterized by variability and it is usually related to the origin of the isolates, as observed in the study. Since E. coli strains isolated from human and poultry face similar challenges in infection establishment of extraintestinal sites, they may share some virulence genes. In this study, most of the 38 genes presented a high frequency in both APEC and UPEC strains. As the distribution of APEC strains in phylogenetic groups showed a significant association with pathogenicity, multiplex-PCR becomes an important tool for screening the pathogenicity of strains isolated from the poultry production chain.