Effects of stocking density on the growth performance and digestive microbiota of broiler chickens.

Increased stocking densities are frequently reported to depress chicken growth performance, but the mechanisms behind this are not fully understood. This study was conducted to investigate the effects of stocking density on growth performance and digestive microbiota, known to be sensitive to environmental factors. Chickens were reared at 2 stocking densities, 12 or 17 birds/m(2). Growth performance was recorded between d 1 and 39, and litter was scored for quality on d 25, 31, and 37. Digestive microbiota was analyzed along the digestive tract (crop, ileum, ceca) of 3- and 6-wk-old chickens by using 2 molecular approaches: a qualitative method (fingerprinting by temporal temperature gradient gel electrophoresis) and a quantitative method (real-time PCR). An increase in stocking density was found to negatively affect the feed conversion ratio (+3.1%) and depress the daily BW gain of broilers (-5.5%) during the period from d 32 to 39 (P ≤ 0.05). Litter quality was reduced with the high stocking density as early as d 25. At 3 wk of age, stocking density strongly affected the fingerprint profiles of the bacterial community, with the highest modifications observed in the crop and ceca (R analysis of similarity = 0.77 and 0.69, respectively, P ≤ 0.05). At 6 wk of age, significant differences in the fingerprint profiles between the stocking densities appeared in the crop and ceca (R analysis of similarity = 0.52 and 0.27, respectively, P ≤ 0.05). The abundance of bacterial groups targeted by real-time PCR was affected by stocking density, but only to a limited extent. Because digestive microbiota may have consequences on the physiology of the digestive tract, its modification by an increase in stocking density may be involved in the reduced growth performance of the bird.

Cytotoxic effect of multinucleation in HeLa cell cultures associated with the presence of Vir plasmid in Escherichia coli strains.

Escherichia coli strain S5 possesses a virulent plasmid termed Vir, which codes for a lethal toxin and a surface antigen. This strain and two trans-conjugant strains, which have received the Vir plasmid from S5, produced a specific and thermolabile cytopathic effect of multinucleation in an HeLa cell cultures assay, whereas isogenic Vir- strains did not. Moreover sonicates of two epidemiologically unrelated Vir+ strains, exerted the same type of cytotoxicity. This effect, together with lethality for chicken, was specifically neutralized by a rabbit antiserum prepared against Vir+ sonicates. The Vir cytopathic effect appeared morphologically distinct from the one caused by the cytotoxic necrotizing factor of E. coli, which was partially related immunologically. We therefore propose to call this type of toxin 'Vir cytotoxin'.

Cytotoxins in non-enterotoxigenic strains of Escherichia coli isolated from feces of diarrheic calves.

We have examined the cytotoxic responses produced in HeLa and Vero cell cultures by sonicates from 15 non-enterotoxigenic (STa-, LT-) strains of E. coli, highly lethal for mice parenterally LD50 less than 3 X 10(7) CFU), which had been isolated from feces of diarrheic calves. Three types of cytotoxic responses were observed. Type 1 (five strains) consisted of enlargement, rounding and polynucleation of HeLa cells, an effect previously reported with cytotoxic necrotizing factor (CNF) in E. coli from infant and piglet enteritis. Type 2 toxicity (three strains and the control Vir strain S5) was also characterized by enlargement and polynucleation of HeLa cells, but in contrast to Type 2 effect, cells were elongated. Sonicates from the latter strains were lethal for chickens, producing the lesions previously described with Vir strains. Type 3 toxicity (two strains and the control VT strain H19), produced an extensive destruction of both Vero and HeLa cell cultures. Cytotoxic effects were completely abolished upon heating for 1 h at 60 degrees C for Type 1 and 2 extracts and at 80 degrees C for Type 3 extracts. Seroneutralization assays showed that cytotoxins of the same type were closely related antigenically. In addition, a slight cross-neutralization was observed between Type 1 (CNF) and Type 2 (Vir) toxins.

Variability in the resistance of four chicken lines to experimental intravenous infection with Salmonella enteritidis phage type 4.

The capacity of four chicken lines (Y11, L2, B13, PA12) to control Salmonella enteritidis (SE) phage type 4 (PT4) systemic colonization was investigated. Thirteen-week-old chickens were intravenously inoculated with 10(6) SE colony-forming units, and the levels of SE colonization were determined at various time intervals after inoculation in liver, spleen, genital organs, and ceca. The course of SE infection showed a rapid contamination of liver, spleen, and genital organs, whereas the ceca were infected later. A significant (P < 0.001) effect of the chicken line on levels of SE was detected on day 3 postinoculation (PI) in liver and ceca, on day 10 PI in ceca, and on day 15 PI in spleen. Because an early control of systemic Salmonella infection by the Ity/Nramp1 gene has been demonstrated in mice, we aimed to study the early resistance of chickens to SE. As a consequence, we then focused our study on the between- and within-line variabilities of SE levels on day 3 PI. According to the SE levels in liver on day 3 PI, the chicken lines could be classified as susceptible (Y11 and L2) or resistant (PA12 and B13). This early variability was explored in resistant B13 and susceptible L2 lines. Differences between these two lines were confirmed in liver but not in ceca. A large within-line variability was observed in all organs of these two lines. The genetic origin of this variability will have to be determined as a prerequisite to an eventual selection.

Experimental study of some factors limiting 'competitive exclusion' of salmonella in chickens.

Some factors affecting the efficiency of competitive exclusion of Salmonella typhimurium var copenhagen in chicks were studied experimentally under gnotobiotic conditions. Axenic chickens were given dilute suspensions of adult faeces and exposed to the salmonellae. Prevention of colonisation of the gut by salmonellae ('competitive exclusion') was variable and depended possibly in part on the source of the adult faeces used to protect the chicks. Exclusion was also dose-dependent, a large inoculum of the salmonellae (10(7) viable organisms per animal) leading to colonisation in treated chicks. An inapparent carrier state was sometimes produced by lower doses of the salmonellae (10(2) viable organisms per animal), but bursts of excretion still appeared after inoculation of the salmonellae. In inapparent healthy carriers, the inoculation of a dose of Eimeria tenella oocysts that was known to produce subclinical caecal coccidiosis led to the shedding of large numbers of salmonellae for over two weeks, and the use of 'competitive exclusion' in poultry as a preventive measure for salmonella infections might thus be limited by the frequent occurrence of subclinical coccidiosis in the field.

[Molecular and epidemiological bases of bacterial resistance to antibiotics]. / Bases moléculaires et épidémiologiques de l'antibiorésistance bactérienne.

The emergence of resistance to antibiotics is related to the genetic determinants of microbial resistance. The characteristics and properties of chromosomal mutations, plasmids, transposons and the various mechanisms of resistance are described. The range of bacterial and genetic exchanges in determining the epidemic spread of resistance to an antibiotic are illustrated and discussed.

[Appearance and evolution of bacterial resistance to antibiotics]. / Apparition et évolution de la résistance bactérienne aux antibiotiques.

Concomitant with antibiotic use has been the appearance of resistance bacteria which seem able to emerge as rapidly as new antibiotics are introduced. Although initially encountered in hospitals, resistant bacteria are now being detected as causes of human and animal infections and as colonizers of the gut. Moreover, resistant bacteria are isolated from the environment.

[In vivo plasmid transfer between strains of Escherichia coli in the chicken digestive system]. / Transfert plasmidique in vivo entre souches d'Escherichia coli dans le tube digestif du poulet.

To study the transfer of plasmids in the gut of chickens, we introduced a conjugative plasmid pGB99, which encodes resistance to chloramphenicol (Cm), tetracycline (Tc), sulphonamide (Su) and trimethoprim (Tm), into a nalidixic acid mutant of an antibiotic sensitive avian E. coli strain (BN118). The transfer of the plasmid between the nalidixic acid resistant strain and a streptomycin resistant mutant of the same strain was studied in the gut of dixenic and gnotobiotic chickens carrying an avian microbial flora devoid of resistant E. coli. Half of the chickens received tetracycline in the drinking water. The results indicate that the plasmid transfer occurred rapidly after inoculation in the gut of the dixenic chickens, with a persistent level of 10(4) transconjugants/g in the feces of chickens without tetracycline. During the administration of tetracycline, selection of the strains harboring the R plasmid (donor strains and transconjugants) was observed. In the gut of the chickens carrying the avian flora plasmid transfer between the donor and recipient strain BN118 was only observed in the group of animals receiving tetracycline. However transfer has probably occurred between the donor strain and the commensal E. coli of the flora in animals of the two groups.

[Presence of plasmids belonging to group incP and conferring chloramphenicol resistance in "Escherichia coli" strains of avian origin (author's transl)]. / Mise en évidence de plasmides appartenant au groupe d'incompatibilité incP conférant la résistance au chloramphénicol chez Escherichia coli d'origine aviaire.

Plasmids pCG76, pCG83, pCG110 and pCG111 were transferred from multiresistant Escherichia coli strains of avian intestinal origin to E. coli K12. They were found to be compatible with reference plasmids of several incompatibility groups, except RP4. This incompatibility with RP4 is a proof that they belong to group incP. Other plasmids of this group were found in E. coli strains isolated from the feces of hens in two different breeding flocks. The incidence and the importance of this plasmid group in the intestinal bacteria of animals are presently investigated.

Inc groups among plasmids harbored by Escherichia coli of avian origin.

The enteric flora of poultry includes a high proportion of antibiotic-resistant Escherichia coli strains harboring conjugative plasmids: 54 of these strains were selected for their ability to transfer tetracycline resistance (associated or not with other resistances). Among the plasmids transferred from these strains, incompatibility tests revealed a high incidence of group Inc I1 and a low frequency of group Inc FII. The importance of group Inc P was confirmed. No Inc N plasmid was found. This pattern differs from what has been reported for human strains.

Spontaneous implantation of antibiotic-resistant Enterobacteriaceae in the digestive tract of chickens in the absence of selective pressure.

In the absence of selective pressure by antibiotics, resistant enterobacteria implanted rapidly in the intestinal tract of chickens, where these organisms subsequently persisted in high numbers. Food could be an important source of this contamination: resistant Escherichia coli present in small numbers in the diet became rapidly and persistently established in the gut. The human caretaker played a passive role in the spread of antibiotic-resistant bacteria between separate groups of chickens. Resistant enteric organisms colonized the gut of animals, with different population sizes. Some strains were able to reach high numbers (10(7) to 10(9)/g), and other strains established themselves at a lower level (10(3) to 10(5)/g), whereas a third type seemed to be only transient inhabitants, unable to persist.

Susceptibility of the rabbit to an enteropathogenic strain of Escherichia coli 0103: effect of animals' age.

Experimental infections with an enteropathogenic strain of Escherichia coli 0103 were studied in SPF rabbits of different ages. Whatever the age, the weight gains in all animals were reduced more or less significantly. However, mortality differed considerably according to the age. Four to 5-week-old rabbits were most susceptible to the infection, with 80-100% mortality associated with diarrhoea, generally of the hemorrhagic type. Peak mortality was observed between 5-10 days post-inoculation. In general, 21-day-old rabbits died from the second week post-inoculation onwards. After the 6th week of age, mortality was rare. Study of the microflora showed that the colonization of the gut by E coli 0103 occurred whatever the age as 10(6)-10(9) bacteria per g of feces of the inoculated strain were recovered. The role of specific receptors has been discussed.

Scanning and transmission electron microscopic study of adherence of Escherichia coli O103 enteropathogenic and/or enterohemorrhagic strain GV in enteric infection in rabbits.

The GV strain (serotype O103:H2:K-), originally isolated from a diarrheic rabbit, is an enteropathogenic Escherichia coli strain that produces diarrhea without synthesizing the classical enterotoxins and that is not invasive. This strain is characterized by a 117-kb plasmid (pREC-1). Histological study of the gut by scanning electron microscopy and transmission electron microscopy was performed on the GV strain, on a derivative strain cured of pREC-1, and on transconjugants obtained by transfer of pREC-1 to nonpathogenic strains E. coli K-12 and 6100, not belonging to the O103 serogroup. The GV strain adhered to the epithelial cells of the ileum and large intestine, whereas the cured GV strain did not. Transfer of plasmid pREC-1 to E. coli K-12 or 6100 allowed the bacteria to attach to the intestinal mucosa in the same manner as that of the wild-type GV strain. Thus, pREC-1 seems to play an important role in attachment to and colonization of the intestinal tract of rabbits by E. coli serogroup O103. Scanning electron microscopy showed numerous bacteria attached together and closely associated with intestinal villi. Transmission electron microscopy revealed effacing lesions characteristic of enteropathogenic E. coli strains: effacing of microvilli and cuplike projections (pedestal formations) associated with an acute inflammatory and hemorrhagic response. In contrast with the results reported for rabbit pathogenic O15 strains, it appeared that the Peyer's patches were not involved in the early stages of infection with the O103 GV strain. This strain may represent a model for the study of the virulence and pathogenic effects of enteropathogenic and enterohemorrhagic E. coli strains.

R plasmid in Escherichia coli O103 coding for colonization of the rabbit intestinal tract.

One rabbit pathogenic Escherichia coli strain, belonging to serogroup O103, harbors a self-transferable 117-kb plasmid (pREC-1) encoding resistance to several antibiotics. The role of this R plasmid in the colonization of the digestive tract in specific-pathogen-free (E. coli O103-free) rabbits was studied. Five-week-old rabbits were inoculated with the wild-type strain, with its variant cured of the plasmid, with an E. coli K-12 strain, or with an untypeable E. coli strain from a healthy rabbit. No symptoms and no mortality were observed in animals inoculated with strains without the plasmid pREC-1, but 87.5% of the rabbits infected by the wild strain died, generally with bloody diarrhea, between days 5 and 15 postinfection. The weight gain of animals was strongly reduced. Transfer of the plasmid to the cured strain or to nonvirulent strains led these strains to induce the same pathology but with a lower mortality. Colonization of the gut by the O103 strain and symptoms of bloody diarrhea are thus related to the presence of the pREC-1 plasmid. The GV strain, which does not produce classical heat-labile enterotoxin or heat-stable enterotoxin and is not invasive, could be considered an enteropathogenic E. coli-like strain. The presence of a conjugative plasmid such as pREC-1 encoding both antibiotic resistance and virulence determinants in O103 E. coli from rabbits could represent a prominent epidemiological hazard under selective pressure by antibiotic therapy.

Parameters controlling interbacterial plasmid spreading in a gnotoxenic chicken gut system: influence of plasmid and bacterial mutations.

Conjugative transfer of R plasmids R64 and R64drd-11 has been compared in vitro and in vivo without selective pressure by antibiotics in a simplified experimental system; the ecosystem was the bowel of germfree chickens, with the host bacteria almost isogenic, and the plasmids differing only in their conjugative transfer frequency. The spread of repressed and derepressed (drd) R plasmids in recipient bacterial populations was very extensive. The repressed phenotype had only a transient effect during the first 4 h. The level of implantation of the donor bacterial population seems to be of minor importance. Only with a poor recipient (con strain) could the spread of R plasmids be reduced and a steady state with a predominantly sensitive bacterial population be established. It is suggested that this steady state results from an equilibrium between the frequencies of R plasmid transfer and loss.

[Quantitative study of capillary and venous bacterial diffusion in experimental bacteremia in chickens]. / Etude quantitative de la diffusion bactérienne capillaire et veineuse lors de bactériémies expérimentales chez le poulet.

Capillary and venous diffusion of bacteria were studied in chickens after intravenous inoculation with Escherichia coli K12. A first group of 35 chickens was inoculated with 10(7) and a second group of 49 chickens with 10(8) E. coli. Bacterial enumeration showed two types of bacterial blood distributions. Early after inoculation, the level of bacteraemia was higher in venous than in capillary blood; later, the level of bacteraemia was higher in capillary than in venous blood. The difference was significant for the two kinds of inoculum. These results showed a slower clearance of E. coli in capillary than in venous blood. These experimental data confirm the greater sensibility of capillary than of venous sampling, already observed in neonates.

Origin and identification of bacteria which produce kairomones in the frass of Acrolepiopsis assectella (Lep., Hyponomeutoidea).

The volatiles used by the parasitoid Diadromus pulchellus to find its host, the leek moth, are produced by the bacteria developing in the frass of the host larvae. The origin and the nature of these bacteria were investigated. Samples were taken from healthy leeks and from infested leeks in the field, as well as from the frass of larvae reared in the laboratory either on the host plant or on an artificial diet. The various species of bacteria identified were cultured in the presence of precursors of leek sulphur volatiles and their volatile emissions were analysed. Klebsiella oxytoca and various Bacillus, common decomposers of plant matter, were the principal species producing active volatiles which were alkyl disulphides.

The role of the O-antigen lipopolysaccharide on the colonization in vivo of the germfree chicken gut by Aeromonas hydrophila serogroup O:34.

We compared the ability of different Aeromonas hydrophila strains from serogroup O:34 grown at different temperatures to colonize in vivo the germfree chicken gut. We found a good colonization when the strains were grown at 20 degrees C but not when they were grown at 37 degrees C. We previously described that these strains were able to form the O-antigen lipopolysaccharide (LPS) when they grow at low temperature but not at high temperature. We also obtained by transposon mutagenesis mutants only devoid of the O-antigen LPS (rfb mutants), and showed that they were unable to colonize the germfree chicken gut. All these results prompted us to conclude that the O-antigen LPS, in these strains, is a main factor for colonization in this animal model system.

[Effect of intravenous human immunoglobulins on bacterial clearance and mortality in experimental Escherichia coli K 1 septicemia in chicken]. / Effet des immunoglobulines humaines intraveineuses sur la clearance bactérienne et la mortalité lors de septicémies expérimentales à Escherichia coli K 1 chez le poulet.

The antibacterial effect of intravenous immune globulins (IVIG) was tested in 4 to 6-week-old chickens experimentally infected with a K1 E. coli strain from a neonate with meningitis. Following inoculation of 10(7) E. coli K1 per animal, the chickens were randomized to receive a placebo, 0.25 g/kg, 0,50 g/kg or 1 g/kg of intravenous immune globulins. Injections were performed 4 hours after the bacterial inoculation. Numbers of animals in each group were 30, 19, 30 and 10 for 6-week-old chickens and 9, 10, 9 and 0 for 4-week-old chickens. Bacterial diffusion was evaluated using quantitative cultures of blood from breast capillaries. In the 6-week-old chickens given 0.25 g/kg or 0.50 g/kg of intravenous immune globulins, bacteremia 48 hours after inoculation and mortality were significantly lower than in the placebo group. Administration of intravenous immune globuline in a dose of 1.0 g/kg, however, was followed by increased blood bacteria counts 48 and 72 hours after inoculation. In the 4-week-old chickens, no reduction in bacterial counts or improvement in survival was seen after administration of intravenous immune globulins. These data should provide a basis for a more rational use of intravenous immune globulins in neonates and children.