Although Pasteurella multocida is a member of the respiratory microbiota, under some circumstances, it is a primary agent of diseases , such as fowl cholera (FC), that cause significant economic losses. Experimental inoculations can be employed to evaluate the pathogenicity of strains, but the results are usually subjective and knowledge on the pathogenesis of this agent is still limited. The objective of this study was to establish a new methodology for classifying the pathogenicity of P. multocida by formulating a standard index. Strains isolated from FC cases and from swine with respiratory problems were selected. One hundred mL of a bacterial culture of each strain, containing 10 6 CFU, was inoculated in 10 one-day-old broilers. Mortality after inoculation, time of death (TD), and the presence of six macroscopic lesions were evaluated over a period of seven days post-inoculation (dpi). A Pathogenicity Index Per Bird (IPI), ranging 0 to 10, was calculated. Liver and heart fragments were collected to reisolate the bacteria. Blood was collected from the surviving birds, and an ELISA test was carried out to detect specific antibodies. The median of the pathogenicity indices, the number of lesions and the rate of bacteria reisolation were significantly different (p<0.05) among the origins of the isolates (p<0.05). The pathogenicity index developed in this study allows the classification of Pasteurella multocida pathogenicity and may be an alternative to the pathogenicity models currently used for screening.
Pasteurella multocida is a Gram-negative bacillus that causes economic losses due to the development of respiratory diseases in several animal species. Among the mechanisms of virulence, the formation of biofilms is an important factor for bacterial survival in hostile environments. Studies of biofilm formation by P. multocida are needed because P. multocida is an important pathogen involved in respiratory infections. However, in contrast to other microorganisms, few studies of biofilm formation have examined P. multocida. Studies comparing the pathogenicity of microbial strains as a function of their biofilm production capacity are also rare. Consequently, the aim of this study was to evaluate the biofilm formation capacity of 94 P. multocida strains isolated from cases of fowl cholera and from swine lungs on polystyrene plates. The associations of the biofilm formation capacity with the pathogenicity index (PI) in vivo and with the presence of four genes (screened by PCR) of the tad locus (tadB, tadD, tadE and tadG), described as adhesion markers, were also determined. Strains from both animal origins were able to form biofilms. However, most of the specimens (52.13%) were classified as weak producers, and more than 40% of the strains of P. multocida (40.42%) did not produce biofilms. There was no significant difference (p>0.05) in the degree of biofilm production between the two sources of isolation. Of the analyzed strains, 56.52% contained all four genes (tadB, tadD, tadE and tadG). The PI arithmetic mean of the strains classified as non-biofilm producers was significantly different (p<0.05) from the PI of moderate-producer strains. The PI of specimens classified as weak biofilm producers also differed significantly (p<0.05) from that of the moderate-producer strains. The results indicate that even though the P. multocida strains isolated from cases of fowl cholera and swine lungs formed biofilms on polystyrene surfaces, adhesion was usually weak. The genes tadB, tadD, tadE and tadG were not significantly associated (p>0.05) with the production of biofilms and with the origin of a given strain. Finally, low virulence strains may suggest a higher biofilm formation capacity on polystyrene plates.
In addition to being the causative agent of fowl cholera (FC), Pasteurella multocida is also one of the most prevalent opportunistic pathogens associated with respiratory diseases in various hosts. However, understanding of the traits that distinguish the virulent isolates that cause FC is still limited. The objective of this study was to characterize P. multocida isolates of Brazil by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis in order to determine if strain-type correlates with virulence or with 22 previously studied virulence genes. The PCR-RFLP was used to classify the isolates into seven strain types, and the isolates in Profile II had a higher pathogenicity index (P < 0.05) than did those in Profiles I, V, and VI. The overall identity among the nucleotide sequences of the ompH was 89.8%. Furthermore, strains available in GenBank showed a high level of homology of the different bacterial serotypes with the groupings resulting from the PCR-RFLP. Strain Types I and II showed the highest identity with Serotypes 3 (100%) and 3-4 (99.1%), respectively. Detection of the pfhA gene indicated the presence of strains that are highly pathogenic. The screening detection of 22 virulence genes and inference through the decision tree models comparing the results of pathogenicity indices permitted the identification of the most highly pathogenic strains of P. multocida .
Fowl cholera is a contagious disease that results from infection by the bacterium Pasteurella multocida. This microorganism is extensively distributed among animal species, but little is known regarding it's pathogenesis and specificity to various hosts. Many studies using pathogenicity evaluation methods are subjective and difficult to quantify because they are often only involved in the observation of the lethal capacity of the agent in experimental inoculation. Due to a lack of more consistent data, this study aimed to establish a classification model of P. multocida pathogenicity in mice using strains isolated from poultry and swine. A total of 94 strains of P. multocida isolated from clinical cases of FC and from lungs of swine were tested. A volume of 0.1 mL of bacterial suspension was obtained from the concentration of 10 6 CFU/mL and inoculated by an intraperitoneal route in five mice. The animals were observed every six hours over seven days. In addition to the mortality observed, the time of death and gross lesions were also analyzed. The Pathogenicity Indexes obtained showed significant differences (p<0.05) according to the origin of the strains. Likewise, the number of gross lesions and isolation percentages were also varied (p<0.05) among strains isolated from poultry and swine. From the observed ratios, the isolates were grouped into three pathogenicity classes: high, medium and low. This study proposed a consistent measurement and classification of P. multocida pathogenicity. The obtained results will be used to generate other adjusted models, as well as to form the basis for disease diagnosis.
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