Laboratory host systems for extraneous agent testing in avian live virus vaccines: problems encountered.

The regulation for batch testing of avian live viral vaccines for extraneous agents was modified recently in order to reduce animal use, and routine batch testing had to be adapted to this new regulation. As a result, however, some tests have become more complicated and time-consuming. Due to systematic technical problems, the new methods required could only be applied to a small proportion of the batches produced. As a consequence, the majority of batches are still tested in animals.

Clonality of Enterococcus faecalis associated with amyloid arthropathy in chickens evaluated by multilocus sequence typing (MLST).

The aim of the study was to evaluate the clonality of 21 strains of Enterococcus faecalis associated with arthritis and amyloid arthropathy by multilocus sequence typing (MLST). The strains originated from five countries. Fifteen of the 21 strains demonstrated the same sequence type, ST82, including a reference amyloid arthropathy strain from the Netherlands. Some of the demonstrated sequence types (ST36, ST59 and ST82) have also been described from human clinical samples while other types are reported for the first time. The results confirm previous observations that outbreaks of amyloid arthropathy seem to be clonally related and indicate a wide distribution of the predominant sequence type which was demonstrated in four countries in Europe and in the USA.

Intestine and environment of the chicken as reservoirs for extraintestinal pathogenic Escherichia coli strains with zoonotic potential.

Although research has increasingly focused on the pathogenesis of avian pathogenic Escherichia coli (APEC) infections and the "APEC pathotype" itself, little is known about the reservoirs of these bacteria. We therefore compared outbreak strains isolated from diseased chickens (n = 121) with nonoutbreak strains, including fecal E. coli strains from clinically healthy chickens (n = 211) and strains from their environment (n = 35) by determining their virulence gene profiles, phylogenetic backgrounds, responses to chicken serum, and in vivo pathogenicities in a chicken infection model. In general, by examining 46 different virulence-associated genes we were able to distinguish the three groups of avian strains, but some specific fecal and environmental isolates had a virulence gene profile that was indistinguishable from that determined for outbreak strains. In addition, a substantial number of phylogenetic EcoR group B2 strains, which are known to include potent human and animal extraintestinal pathogenic E. coli (ExPEC) strains, were identified among the APEC strains (44.5%) as well as among the fecal E. coli strains from clinically healthy chickens (23.2%). Comparably high percentages (79.2 to 89.3%) of serum-resistant strains were identified for all three groups of strains tested, bringing into question the usefulness of this phenotype as a principal marker for extraintestinal virulence. Intratracheal infection of 5-week-old chickens corroborated the pathogenicity of a number of nonoutbreak strains. Multilocus sequence typing data revealed that most strains that were virulent in chicken infection experiments belonged to sequence types that are almost exclusively associated with extraintestinal diseases not only in birds but also in humans, like septicemia, urinary tract infection, and newborn meningitis, supporting the hypothesis that not the ecohabitat but the phylogeny of E. coli strains determines virulence. These data provide strong evidence for an avian intestinal reservoir hypothesis which could be used to develop intestinal intervention strategies. These strains pose a zoonotic risk because either they could be transferred directly from birds to humans or they could serve as a genetic pool for ExPEC strains.

The chicken as a natural model for extraintestinal infections caused by avian pathogenic Escherichia coli (APEC).

E. coli infections in avian species have become an economic threat to the poultry industry worldwide. Several factors have been associated with the virulence of E. coli in avian hosts, but no specific virulence gene has been identified as being entirely responsible for the pathogenicity of avian pathogenic E. coli (APEC). Needless to say, the chicken would serve as the best model organism for unravelling the pathogenic mechanisms of APEC, an extraintestinal pathogen. Five-week-old white leghorn SPF chickens were infected intra-tracheally with a well characterized APEC field strain IMT5155 (O2:K1:H5) using different doses corresponding to the respective models of infection established, that is, the lung colonization model allowing re-isolation of bacteria only from the lung but not from other internal organs, and the systemic infection model. These two models represent the crucial steps in the pathogenesis of APEC infections, including the colonization of the lung epithelium and the spread of bacteria throughout the bloodstream. The read-out system includes a clinical score, pathomorphological changes and bacterial load determination. The lung colonization model has been established and described for the first time in this study, in addition to a comprehensive account of a systemic infection model which enables the study of severe extraintestinal pathogenic E. coli (ExPEC) infections. These in vivo models enable the application of various molecular approaches to study host-pathogen interactions more closely. The most important application of such genetic manipulation techniques is the identification of genes required for extraintestinal virulence, as well as host genes involved in immunity in vivo. The knowledge obtained from these studies serves the dual purpose of shedding light on the nature of virulence itself, as well as providing a route for rational attenuation of the pathogen for vaccine construction, a measure by which extraintestinal infections, including those caused by APEC, could eventually be controlled and prevented in the field.

Avian pathogenic, uropathogenic, and newborn meningitis-causing Escherichia coli: how closely related are they?

Avian pathogenic Escherichia coli (APEC), uropathogenic E. coli (UPEC), and newborn meningitis-causing E. coli (NMEC) establish infections in extraintestinal habitats (extraintestinal pathogenic E. coli; ExPEC) of different hosts. As diversity, epidemiological sources, and evolutionary origins of ExPEC are so far only partially defined, we screened a collection of 526 strains of medical and veterinary origin of various O-types for assignment to E. coli reference collection (ECOR) group and virulence gene patterns. Results of ECOR typing confirmed that human ExPEC strains mostly belong to groups B2, followed by group D. Although a considerable portion of APEC strains did also fell into ECOR group B2 (35.1%), a higher amount (46.1%) belonged to group A, which has previously been described to also harbour strains with a high pathogenic potential for humans. The number of virulence-associated genes of single strains ranged from 5 to 26 among 33 genes tested and high numbers were rather related to K1-positive and ECOR B2 strains than to a certain pathotype. With a few exceptions (iha, afa/draB, sfa/foc, and hlyA), which were rarely present in APEC strains, most chromosomally located genes were widely distributed among all ExPEC strains irrespective of host and pathotype. However, prevalence of invasion genes (ibeA and gimB) and K1 capsule-encoding gene neuC indicated a closer relationship between APEC and NMEC strains. Genes associated with ColV plasmids (tsh, iss, and the episomal sit locus) were in general more prevalent in APEC than in UPEC and NMEC strains, indicating that APEC could be a source of ColV-located genes or complete plasmids for other ExPEC strains. Our data support the hypothesis that (a) poultry may be a vehicle or even a reservoir for human ExPEC strains, (b) APEC potentially serve as a reservoir of virulence-associated genes for UPEC and NMEC, (c) some ExPEC strains, although of different pathotypes, may share common ancestors, and (d) as a conclusion certain APEC subgroups have to be considered potential zoonotic agents. The finding of different evolutionary clusters within these three pathotypes implicates an independently and parallel evolution, which should be resolved in the future by thorough phylogenetic typing.

Investigations of the vertical transmission of Erysipelothrix rhusiopathiae in laying hens.

Erysipelas was diagnosed in a layer breeder flock in Sweden in 2002. Although vertical transmission of Erysipelothrix rhusiopathiae has not been previously described in chickens, the potential of erysipelas infection to adversely affect hatching eggs was of concern. To clarify the possible impact of erysipelas on hatching eggs and their progeny, an experiment was done using 200 hatching eggs collected from the infected flock. The eggs were incubated for 21 days, and the egg shells, infertile eggs, dead-in-shell embryos, and a sample of day-old hatched chicks and blood samples from 5-day-old chicks were cultured for E. rhusiopathiae. In addition, after 28 days of grow-out, the male chickens were euthanatized and cultured for the bacterium, and the remaining female chickens were placed as a backyard flock and observed over a 4-mo period. Bacteriological test results of the above-mentioned samples were negative for E. rhusiopathiae. Mortality rates were not excessive, and no clinical symptoms of erysipelas were observed during the period of observation. The result of the investigation suggests that in layer breeder chickens, E. rhusiopathiae is not vertically or egg transmitted and that the disease outbreak in the parent stock had no adverse impact on the quality of hatching eggs in terms of increased embryo mortality.

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.

Molecular epidemiology of avian pathogenic Escherichia coli (APEC) isolated from colisepticemia in poultry.

The molecular biology and epidemiology of 150 avian pathogenic Escherichia coli strains (APEC) isolated from septicemic poultry in Germany was investigated by serotyping, pulsed field gel electrophoresis (PFGE), and polymerase chain reaction (PCR). Only 49.6% of the isolates could be grouped to serogroups O1, O2, and O78. Macrorestriction analyses data revealed two large clonal groups (clusters I and II) among the APEC strains with a similarity of 60.9% to each other. An association between restriction pattern and serogroup or origin of the strains was only present in a few subgroups of each clusters I and II, but was not evident. In contrast, our data revealed distinct combinations of virulence-associated genes in that 51.2% of the O2-strains harboured a combination of the genes fyuA, irp2, iucD, tsh, vat, fimC, and colV and 36.4% of the O78-strains possessed the same gene combination with exception of vat. With 34 different gene combinations the non-O1, -O2, -O78 isolates revealed a higher variability in their virulence gene pattern than O1-, O2-, and O78-strains with 6, 13, and 9 patterns, respectively. Our data indicate only a limited association between the virulence gene pattern and the serogroup of APEC strains and question the sensitivity of O-typing for APEC identification without the application of further diagnostic tools. Although a limited number of APEC clones exist, horizontal gene transfer seems to be common in these pathogens. These findings strengthen further research on the population structure of APEC and may be the reason for the lack of clear definition of this common E. coli pathotype.