Rapid detection of virulence-associated genes in avian pathogenic Escherichia coli by multiplex polymerase chain reaction

Based on recently published prevalence data of virulence-associated factors in avian pathogenic Escherichia coli (APEC) and their roles in the pathogenesis of colibacillosis, we developed a multiplex polymerase chain reaction (PCR) as a molecular tool supplementing current diagnostic schemes that mainly rely on serological examination of strains isolated from diseased birds. Multiple isolates of E. coli from clinical cases of colibacillosis known to possess different combinations of eight genes were used as sources of template DNA to develop the multiplex PCR protocol, targeting genes for P-fimbriae (papC), aerobactin (iucD), iron-repressible protein (irp2), temperature-sensitive hemagglutinin (tsh), vacuolating autotransporter toxin (vat), enteroaggregative toxin (astA), increased serum survival protein (iss), and colicin V plasmid operon genes (cva/cvi). In order to verify the usefulness of this diagnostic tool, E. coli strains isolated from fecal samples of clinically healthy chickens were also included in this study, as were uropathogenic (UPEC), necrotoxigenic, and diarrhegenic E. coli strains. The application of the multiplex PCR protocol to 14 E. coli strains isolated from septicemic poultry showed that these strains harbored four to eight of the genes mentioned above. In contrast, those isolates that have been shown to be nonpathogenic for 5-wk-old chickens possessed either none or, at most, three of these genes. We found only one enterohemorrhagic (EHEC), one enteropathogenic (EPEC), and two enterotoxic (ETEC) E. coli strains positive for irp2, and another two ETEC strains positive for astA. As expected, UPEC isolates yielded different combinations of the genes iss, papC, iucD, irp2, and a sequence similar to vat. However, neither the colicin V operon genes cva/cvi nor tsh were amplified in UPEC isolates. The multiplex PCR results were compared with those obtained by DNA-DNA-hybridization analyses to validate the specificity of oligonucleotide primers, and the protocol was concluded to be a useful, sensitive, and rapid assay system to detect avian pathogenic E. coli and differentiate them from nonpathogenic strains and those belonging to other pathotypes.     

Avian pathogenic Escherichia coli (APEC)

Infections with avian pathogenic Escherichia coli (APEC) cause colibacillosis, an acute and mostly systemic disease resulting in significant economic losses in poultry industry worldwide. Avian colibacillosis is a complex syndrome characterized by multiple organ lesions with airsacculitis and associated pericarditis, perihepatitis and peritonitis being most typical. Environmental factors as well as the constitution of poultry or initial viral infections influence the outcome of APEC-infections. However, several challenge experiments in chickens proofed the role of virulent APEC strains as the single aetiological agent. Currently serotypes O1:K1, O2:K1 and O78:K80 are recognized as the most prevalent, however the number of published serotypes is increasing. In addition, single APEC isolates vary profoundly in virulence, and knowledge about the molecular basis of this variability is still scarce. Known virulence factors of APEC are adhesins (F1- and P-fimbriae), iron acquisition systems (aerobactin and yersiniabactin), hemolysins (hemolysinE and temperaturesensitive hemagglutinin), resistance to the bactericidal effects of serum and phagocytosis (outer membrane protein, iss protein, lipopolysaccharide, K/1)-capsule and colilcin production) as well as toxins and cytotoxins (heat stable toxin, cyto-/verotoxin and flagella toxin). Esperimental studies have shown that the respiratory tract, principally the gas-exchange region of the lung and the interstitium of the air sacs are the most important sites of entry for avian pathogenic E. coli. APEC strains adhere to the epithelial cells of air sacs presumably through F1-fimbriae. After colonization and multiplication the bacteria enter the bloodstream, and the temperature-sensitive hemagglutinin (tsh) seems to be important int his step. After invading the bloodstream APEC cause a septicemia resulting in massive lesins in multiple internal organs and in sudden death of the birds. The ability of the bacteria to acquire iron and the resistance to the bactericidal effects of serum, predominantly conferred by the increased serum survival (iss)--protein, enables APEC to multiply quickly in their hosts. Iss is regarded a specific genetic marker for avian pathogenic E. colistrains. A critical review of the literature published so far on APEC reveals, that these pathotypes are not defined appropriately. This findings urge investigations on the population structure of APEC, enabling the establishment of appropriate diagnostic tools and avoiding the obsolete use of serotyping for APEC diagnosis. So far more than 20 APEC strains have been investigated in animal experiments, explaining contrary published results. Thus, the lack of knowledge in pathogenicity and in immunity of APEC infections urges further experimental studies. As APEC share not only identical serotypes with human pathogens but also specific virulence factors, their zoonotic potential is under consideration.     

Comparative quantification of the in vitro activity of veterinary fluoroquinolones

The aim of this study was to compare the in vitro antimicrobial activity of the veterinary fluoroquinolones against a panel of recently isolated porcine and bovine bacterial pathogens. The study used enrofloxacin as a benchmark against which other agents were compared, being the most common fluoroquinolone used in treatment of bovine and porcine infections. The activity of ciprofloxacin was also assessed as it is the main metabolite of enrofloxacin in cattle. Enrofloxacin and ciprofloxacin generally showed higher antibacterial activity, in terms of MIC(50) values, for most pathogen species when compared with marbofloxacin, difloxacin, danofloxacin and norfloxacin. Ciprofloxacin showed significantly greater in vitro antibacterial activity than enrofloxacin against M. haemolytica, P. multocida and E. coli, whereas enrofloxacin showed greater activity than ciprofloxacin against S. aureus. Marbofloxacin was significantly more active than enrofloxacin against M. haemolytica, E. coli and B. bronchiseptica but less active against P. multocida, S. aureus, coagulase negative Staphylococci, S. dysgalactiae, S. uberis, A. pleuropneumoniae and S. suis. Danofloxacin was significantly less active than enrofloxacin against P. multocida, E. coli, S. uberis, A. pleuropneumoniae and S. suis. Enrofloxacin and its metabolite ciprofloxacin showed the highest in vitro activities against most bovine pathogens tested and the porcine pathogens also showed a high degree of sensitivity to enrofloxacin. These data facilitate further pharmacokinetic/pharmacodynamic comparison of fluoroquinolones currently used in veterinary medicine.     

The Interdependence of Deformational and Thermal Processes in Mountain Belts

Crustal temperatures within collisional orogens are anomalously high compared with temperatures at comparable depths in stable continents, which is evidence of thermal processes that are fundamental to orogenesis. These temperatures can be explained by the redistribution of crust enriched in heat-producing elements through the accretion of crust from the down-going plate to the upper plate and surface erosion. With the use of geologically reasonable rates, the model results predict high temperatures (over 600°C) and inverted upper-plate geotherms (about 100°C over 20 kilometers) at shallow depths (20 to 40 kilometers) by 25 to 35 million years after collision. This study emphasizes the interdependence of deformational, surficial, and thermal processes.     

Role of Gene Interactions in Hybrid Speciation: Evidence from Ancient and Experimental Hybrids

The origin of a new diploid species by means of hybridization requires the successful merger of differentiated parental species' genomes. To study this process, the genomic composition of three experimentally synthesized hybrid lineages was compared with that of an ancient hybrid species. The genomic composition of the synthesized and ancient hybrids was concordant (rs = 0.68, P < 0.0001), indicating that selection to a large extent governs hybrid species formation. Further, nonrandom rates of introgression and significant associations among unlinked markers in each of the three synthesized hybrid lineages imply that interactions between coadapted parental species' genes constrain the genomic composition of hybrid species.     

Studies on the artificial propagation of Monopterus albus (Zuiew)

Artificial propagation of Monopterus albus (Zuiew) was conducted during their spawning season in the present study. Different doses of luteinizing-hormone-releasing-hormone analogue (LHRH-A) were tested for inducing the ovulation of brood females. No significant difference was found in the degree-hours and fertilization rates between three different doses (0.1, 0.2 and 0.3 μg LHRH-A g^-1 fish). A fertilization solution of 0.3% NaCl produced significantly higher fertilization rates than the other three solutions (0.5% NaCl, 0.65% NaCl and water). Fertilization times ranging from 5 to 2 0 min gave no significant difference in the fertilization rates. The embryonic and larval development of M. albus was observed and illustrated. The hatching time of eggs of M. albus was about 140 h in the 28-3 0^oC water temperature range. Using Tubifex spp. for the first food for the larval nursery gave higher growth rate than two other foods, zooplankton and artificial food.