Effects of metals and persistent organic pollutants on the fitness and health of juveniles of the endangered european sturgeon Acipenser sturio Exposed to W1ater and sediments of the garonne and dordogne rivers.

The last remaining population of European sturgeon (Acipenser sturio) lives in the Gironde-Garonne-Dordogne (France) catchment (GGD). Captive young individuals are released into the GGD hydrosystem each year, as part of a restocking programme. This study aims to assess the health status of juveniles A. sturio to current conditions in the GGD hydrosystem, to evaluate their capacity to survive and grow in a moderately anthropized ecosystems. 3-month-old farmed sturgeons were exposed for one month in experimental conditions that mimic the environmental conditions in the Garonne and Dordogne rivers, followed by five months of depuration. After one month of exposure, fish exposed to Dordogne and Garonne waters bioaccumulated higher levels of metals and persistent organic pollutants, displayed a reduced hepato-somatic index, and had depleted levels of lipids and glycogen content in their liver, when compared with the Reference group. However, metabolic and swimming performance, as well as the costs of swimming were not impaired. After the 5 months depuration, a significant decrease of K was observed for all exposure conditions. HSI also decreased with time. The overall health status and adaptive capacity of juvenile A. sturio appeared to be maintained over the experimental 6 months' period. Juveniles of A. sturio seem to have the adaptive capacity to survive and grow in the GGD hydrosystem, after being released as part of a restocking programme.

Nation-wide monitoring campaign of 49 biocides and surfactants in surface waters and wastewaters.

Despite their intensive use and their impact on ecosystems, biocides and surfactants are still poorly regulated and poorly monitored at large scale. In the frame of the revision of the national regulatory surveillance plan of surface waters, France planned in 2018 a monitoring campaign at national scale focused on these two types of substances of very emerging concern. Forty-nine contaminants (32 biocides and 17 surfactants) were investigated in surface water and sediment samples from 91 sampling sites, and in effluent and sludge samples of 7 wastewater treatment plants (WWTP), in mainland France and overseas regions. Between 33 and 52 % of the target contaminants were quantified at least once in water and sediment. High frequencies of quantification were observed for the surfactants (up to 91 % in water samples and up to 57 % in sediment samples for LAS C10-C13) and for the biocides (up to 64 % for fipronil in water samples and up to 90 % for methyl nonyl ketone in sediment samples). The median concentrations of surfactants were up to 2 µg/L in mainland surface water samples and up to 528 µg/kg in sediment samples, and for biocides, the median concentrations were up to 0.18 µg/L in mainland surface water samples and up to 104 µg/kg in sediment samples. PNEC exceedances in water and sediment were determined for both types of substances. The analysis of effluent and sludge suggested significant but not total removal of these substances in the WWTP. Temporal and spatial variations of the concentrations of both types of substances in surface water samples were also observed, suggesting both punctual and diffuse contamination sources of the surface water investigated.

Physiological effects of PFAS exposure in seabird chicks: A multi-species study of thyroid hormone triiodothyronine, body condition and telomere length in South Western France.

There is growing evidence that poly and perfluoroalkyl substances (PFAS) exposure leads to the disruption of thyroid hormones including thyroxine (T4) and triiodothyronine (T3), and may affect telomeres, repetitive nucleotide sequences which protect chromosome ends. Many seabird species are long-lived top predators thus exhibit high contaminant levels, and PFAS-disrupting effects on their physiology have been documented especially in relation to the endocrine system in adults. On the contrary, studies on the developmental period (i.e., chicks), during which exposure to environmental contaminants may have a greater impact on physiological traits, remain scarce to this date. We carried out a multi-species study with the aim to assess whether and to which extent chicks of four gull species (herring gull, great and lesser black-backed gull, yellow-legged gull) in South Western France are contaminated by PFAS, and to bring further evidence about their potential physiological consequences. Linear PFOS showed concentrations of concern as it was generally >10 times higher than the other PFAS, and exceeded a threshold toxicity level (calculated from previous studies in birds) in almost all sampled chicks. Nonetheless, in herring gull male chicks, total T3 levels were significantly and negatively associated with perfluorodecanoate (PFDA) and perfluorododecanoate (PFDoDA) and positively associated with perfluorotetradecanoate (PFTeDA) in female chicks. Total T3 levels were also positively associated with PFDoDA in great black backed gull male chicks and with perfluorotridecanoate (PFTrDA) in lesser black backed gull chicks. In lesser and great black-backed gulls, both females and males showed significant negative associations between several PFAS and their body condition, and a positive association between telomere length and L-PFOS in the yellow-legged gull was also found. These results corroborate previous findings and need to be further explored as they suggest that PFAS may interfere with the physiological status of chicks during the developmental period, potentially inducing long-lasting consequences.

High levels of fluoroalkyl substances and potential disruption of thyroid hormones in three gull species from South Western France.

Per- and poly-fluoroalkyl substances (PFAS) raised increasing concerns over the past years due to their persistence and global distribution. Understanding their occurrence in the environment and their disruptive effect on the physiology of humans and wildlife remains a major challenge in ecotoxicological studies. Here, we investigate the occurrence of several carboxylic and sulfonic PFAS in 105 individuals of three seabird species (27 great black-backed gull Larus marinus; 44 lesser black-backed gull Larus fuscus graellsii; and 34 European herring gull Larus argentatus) from South western France. We further estimated the relationship between plasma concentrations of PFAS and i) the body condition of the birds and ii) plasma concentrations of thyroid hormone triiodothyronine (TT3). We found that great and lesser black-backed gulls from South Western France are exposed to PFAS levels comparable to highly contaminated species from other geographical areas, although major emission sources (i.e. related to industrial activities) are absent in the region. We additionally found that PFAS are negatively associated with the body condition of the birds in two of the studied species, and that these results are sex-dependent. Finally, we found positive associations between exposure to PFAS and TT3 in the great black-backed gull, suggesting a potential disrupting mechanism of PFAS exposure. Although only three years of data have been collected, we investigated PFAS trend over the study period, and found that great black-backed gulls document an increasing trend of plasma PFAS concentration from 2016 to 2018. Because PFAS might have detrimental effects on birds, French seabird populations should be monitored since an increase of PFAS exposure may impact on population viability both in the short- and long-term.

Combined spatial and retrospective analysis of fluoroalkyl chemicals in fluvial sediments reveal changes in levels and patterns over the last 40 years.

Bed sediments and a dated sediment core were collected upstream and downstream from the city of Lyon (France) to assess the spatial and temporal trends of contamination by per- and polyfluoroalkyl substances (PFASs) in this section of the Rhône River. Upstream from Lyon, concentrations of total PFASs (ΣPFASs) in sediments are low (between 0.19 and 2.6 ng g-1 dry weight - dw), being characterized by a high proportion of perfluorooctane sulfonate (PFOS). Downstream from Lyon, and also from a fluoropolymer manufacturing plant, ΣPFASs concentrations reach 48.7 ng g-1 dw. A gradual decrease of concentrations is reported at the coring site further downstream (38 km). Based on a dated sediment core, the temporal evolution of PFASs is reconstructed from 1984 to 2013. Prior to 1987, ΣPFASs concentrations were low (≤2 ng g-1 dw), increasing to a maximum of 51 ng g-1 dw in the 1990s and then decreasing from 2002 to the present day (∼10 ng g-1 dw). In terms of the PFAS pattern, the proportion of perfluoroalkyl sulfonic acids (PFSAs) has remained stable since the 1980s (∼10%), whereas large variations are reported for carboxylic acids (PFCAs). Long chain- (C > 8) PFCAs characterized by an even number of perfluorinated carbons represent about 74% of the total PFAS load until 2005. However, from 2005 to 2013, the relative contribution of long chain- (C > 8) PFCAs with an odd number of perfluorinated carbons reaches 80%. Such changes in the PFAS pattern likely highlight a major shift in the industrial production process. This spatial and retrospective study provides valuable insights into the long-term contamination patterns of PFAS chemicals in river basins impacted by both urban and industrial activities.

Automated image analysis of bacterial colony growth as a tool to study individual lag time distributions of immobilized cells.

A method to determine the individual lag time (lag) distributions of immobilized bacteria was presented. The method was based on the image analysis of the bacterial colony growth. The lag distributions were retrieved from the distributions of the detection times (Td) required to form macroscopically visible colonies. Using this method, the lag distributions on agar for Listeria monocytogenes cells previously subjected to two situations reproducing conditions encountered during the contamination of cheese, were determined. The results were presented and compared with lag distributions obtained with an established method based on the time to detection of turbidity in broth. An original method to retrieve lag in broth and agar without any knowledge of the growth rate was also proposed. In order not to bias the distributions of lag on agar the impact of spatial separation between colonies on colony growth rates was quantified. Means and standard deviations of lag distributions for the two different stresses were found to be similar in broth and on agar. Extreme Value type II distributions fitted the best the different datasets of lag distributions.

Recruitment of 99m-technetium- or 111-indium-labelled polymorphonuclear leucocytes in experimentally induced pyogranulomas in lambs.

The recruitment of polymorphonuclear leucocytes (PMNs) during the development of experimental pyogranulomas induced by Corynebacterium pseudotuberculosis was followed in nine male lambs by scintigraphic examination. Autologous blood PMNs were labelled with 99m-technetium or 111-indium and were re-injected intravenously into infected lambs. The functional properties of the labelled cells were monitored 1) in vitro by measuring their phagocytic and bactericidal activity against C. pseudotuberculosis and their chemotaxis under agarose, and 2) in vivo by following scintigraphically their capacity to accumulate in an inflammatory focus induced by intradermal injection of latex beads coated with Salmonella abortus equi lipopolysaccharide. Following inoculation of corynebacteria into the right ear of lambs, radioactive foci were observed to be localized in the right ear and in the draining lymph nodes during the 4 days following inoculation. Histopathological examination performed 32 h after inoculation confirmed the intense accumulation of PMNs at these sites. With the exception of one animal, which presented visible foci in the neck 14 days postinoculation, no radioactive foci were observed during the later phases of experimental infection, despite the presence of multiple pyogranulomas which were confirmed by bacteriological examination after necropsy of the lambs. Histopathological examination of these lesions revealed layers of fibroblasts, lymphocytes, and macrophages surrounding a necrotic centre. The results of these studies suggest that the contribution of PMNs during the chronic phase of inflammation is considerably reduced in comparison with the acute inflammatory phase of the infectious process.

Epithelioid and fibroblastic cell lines derived from the ileum of an adult histocompatible miniature boar (d/d haplotype) and immortalized by SV40 plasmid.

Intestinal explants were maintained for weeks in a growth medium containing collagenase for progressive digestion to derive finite cell lines from the ileum (64 lines) or from the colon (8 lines) of a boar. Two ileal cell lines retaining either a fibroblastic or an epithelioid morphology have been used to derive heteroploid cell lines (IPI-1 and IPI-2) immortalized by transfection with an SV40 plasmid (pSV3-neo). The IPI-1 cells were found of fibroblastic lineage. The IPI-2 cell line gave rise to morphologically heterogeneous colonies ranging from typical epithelial cells to colonies of more-elongated cells. A crisis occurred during subcultivation of IPI-2 leading to the isolation of the IPI-2I cell line with a 24 h doubling time and a 21% plating efficiency. Epithelial nature of IPI-2I cells was supported by ultrastructural analysis of the cell monolayers. Differentiated cells were found to express microvilli at the apical cellular membrane and desmosomes connecting adjacent cells. Stable epithelioid phenotypes were obtained only from the IPI-2I cell line by multiple subcloning. These cells were found to express characteristics of both epithelial and mesenchymal cells by positive immunostaining with monoclonal antibodies reacting either with keratin 18 filament of simple epithelia or with vimentin filament typical in vivo of mesoderm. The lack of villin expression and the absence of transepithelial resistance have to be related to a poor differentiated state of this cell line. All these immortalized cell lines were permissive to the replication of microorganisms pathogenic for pig (Salmonella chloleraesuis, Salmonella typhimurium and tissue culture-adapted strains of transmissible gastroenteritis virus). The collection of finite and continuous cell lines will help to develop in vitro methods for long-term propagation of freshly isolated epithelium or three-dimensional organ culture in pig. In addition, the IPI-2I cell line provides a new model to study the conversion from a transformed to a nontransformed phenotype as incorporation of 2% dimethyl sulfoxide in the growth medium to repress large tumor antigen expression led to the progressive disappearance of cytokeratin 18 positive cells with, over a week, the death of the surviving vimentin-positive cells.

Salmonella typhimurium TnphoA mutants with increased sensitivity to biological and chemical detergents.

Salmonella typhimurium is a ubiquitous pathogenic bacterium able to sustain the environmental conditions of the gastrointestinal tract, including biliary salts. To understand the mechanisms involved in bile salt resistance and, more generally, detergent resistance, we investigated S. typhimurium mutants produced with the random mutagenic TnphoA transposon. A total of 3,000 transpositional mutants were isolated. Three strains among the 1,432 first mutants lost the ability to grow in the presence of biological and chemical detergents. They were prototrophic and exhibited normal lipopolysaccharide and outer membrane protein profiles after SDS-PAGE. They did not show sensitivity to dyes but showed very different sensitivities to antibiotics. For each mutant strain, Southern blotting analysis revealed a unique TnphoA insertion at different chromosomal locations. These observations were confirmed by transduction experiments.

Experimental ovine salmonellosis (Salmonella abortusovis): pathogenesis and vaccination.

Salmonella enterica subsp. enterica ser. Abortusovis, a sheep-adapted serotype, causes a contagious disease. Abortion is the major symptom and the main source of contamination. Research on this ovine disease may aid farmers, but may also contribute to comparative biological knowledge. Innate resistance partly controlled by the Ity locus, increased resistance to reinfection and humoral and T-cell-mediated immunity were observations gained with a murine model. In ewes, abortion regularly occurs following subcutaneous challenge carried out from the third month of gestation onwards. This ovine model was used to evaluate prevention methods for Salmonella Abortusovis infection. One subcutaneous injection of a live attenuated lyophilized vaccine containing a selected streptomycin-independent reverse mutant was shown to protect ewes against abortion and excretion of Salmonella Abortusovis. This vaccine could be administered simultaneously with other commercial live vaccines such as Brucella melitensis Rev. 1 vaccine. In sheep, application of the vaccine to the conjunctiva (an easy, individual and hygienic route of mucosal vaccination) was followed by lymph node bacterial colonization and a serological response without local or general clinical reactions. The early events of natural infection remain to be explored, as do the mechanisms underlying the host specificity of Salmonella Abortusovis.

Identification of a new locus in Listeria monocytogenes involved in cellobiose-dependent repression of hly expression.

Expression of the PrfA-controlled virulence gene hly (encoding the pore-forming cytolysin listeriolysin) is down-regulated by readily metabolized carbon sources in Listeria monocytogenes. We isolated a Tn917-insertional mutant of L. monocytogenes (strain LO28), which expressed a hemolytic phenotype in the presence of cellobiose. Using hly fusions to luxAluxB genes, we show that hly expression was derepressed in the presence of cellobiose at the transcriptional level. Surprisingly, hly expression was still repressed by glucose, as observed for the parental strain. Genetic analysis of the Tn917-flanking regions indicated that the transposon had inserted in a non-coding region located between two genes in opposite orientations. These two newly identified genes were designated orfA and mdrL. The insertion occurred immediately upstream of orfA, likely into its promoter region. Transcriptional analysis of orfA and mdrL revealed that Tn917 had abolished orfA expression whereas it had activated expression of mdrL. orfA encodes a putative protein of 176 amino acids homologous to YfiO of Bacillus subtilis (28% identity), a protein of unknown function. mdrL codes for a putative protein of 398 amino acids homologous to Bmr and Blt of B. subtilis (21-24% identity), two members of the multidrug resistance efflux pump family. Our results indicate that we have identified a new locus which plays a crucial role in the cellobiose-dependent repression of hly expression.

Salmonella typhimurium acrB-like gene: identification and role in resistance to biliary salts and detergents and in murine infection.

Salmonella serotype typhimurium transpositional mutants altered in resistance to biliary salts and detergents were isolated previously. We have characterized further the LX1054 mutant strain, the most sensitive of them. The chromosomal DNA segment flanking transposon insertion was cloned and sequenced. The highest level of identity was found for the acrB (formerly acrE) gene of Escherichia coli, a gene encoding a drug efflux pump of the Acr family. LX1054 exhibited a reduced capacity to colonize the intestinal tract. After passages in mice, the mutant strain lost the sensitive phenotype. In vitro, a resumption of growth appeared after 17 h of culture in medium with cholate or other tested biological or chemical detergents. Then, the acquired resistant phenotype seemed stable. The data suggested a role of S. typhimurium acrB-like gene in resistance to biliary salts and detergents and in mice intestinal colonization. However, the local and transient sensitivity observed in vivo, and the in vitro adaptations suggest that several detergent-resistance mechanisms operate in S. typhimurium.

Assessment of the virulence of Listeria monocytogenes: agreement between a plaque-forming assay with HT-29 cells and infection of immunocompetent mice.

Some Listeria monocytogenes strains not related to clinical cases have been found to exhibit a low virulence level in mice as well as in an in vitro test using Caco-2 cells. The purpose of this study was to validate a new in vitro test of virulence based on a plaque-forming assay (PFA) using a HT-29 cell monolayer with 118 Listeria strains. The use of HT-29 cells in 96-well tissue culture plates allowed the testing of 30 strains per day and providing results in 24 h. In addition. statistical analyses demonstrated the reproducibility and repeatability of the PFA. No quantitative relationship was observed between the virulence of the strains and the hemolytic titer or the cytotoxic effects on HT-29 cells. In contrast, good agreement was observed between virulence assessed after subcutaneous (SC) infection and virulence obtained by PFA. Three groups of L. monocytogenes strains (avirulent, hypovirulent and fully virulent) were established by comparison of the clinical origin of the strains, the number of immunocompetent contaminated mice and the numbers of Listeria strains recovered in the spleen after SC infection. With one exception, i.e. a clinical case of L. seeligeri (sensitivity 0.98), the PFA successfully detected the virulent strains only (specificity 1). Decision-tree algorithms performed by SAS and S-Plus demonstrated that this tissue culture assay discriminated between the avirulent and hypovirulent strains and the virulent strains. This test could therefore be an alternative to in vivo tests, allowing grading of virulence.

Comparative histopathology of draining lymph node after infection with virulent or attenuated strains of Salmonella abortusovis in lambs.

Histological responses to the early phase of infection were compared in parotid lymph nodes of lambs infected by the subcutaneous route into the right eyelid with either a virulent or an attenuated strain of Salmonella abortusovis. The right parotid lymph nodes showed a massive PMN infiltration for the first days of infection for both strains. From day 6, the infected lymph nodes developed a lymphoid hyperplasia with prominent germinal centers independent of strain type. The virulent strain of S. abortusovis induced focal lesions in 2 out of 6 lambs necropsied on days 6 and 10, and provoked a systemic infection evidenced by the regular colonization of spleen on day 6. In contrast, no focal lesion and a restricted bacterial dissemination were observed in lambs infected with the vaccinal strain.

Histopathology of the early phase during experimental Corynebacterium pseudotuberculosis infection in lambs.

Histological responses during experimental Corynebacterium pseudotuberculosis infection in lambs were investigated in parotid lymph nodes for ten days following inoculation. Lambs were infected by the subcutaneous route into the right eyelid with a virulent strain of C. pseudotuberculosis. Multiple microscopic acute abscesses, predominantly infiltrated with polymorphonuclear (PMN) leucocytes, were seen in the right parotid lymph node on the 1st day post-inoculation (PI). This massive PMN infiltration coincided with a peripheral blood granulocytosis. On day 3 PI, an influx of histiocytes was observed, while the microabscesses became confluent. From day 3 to day 10 PI, these lesions became enlarged and transformed into typical pyogranulomas with a central necrosis and a peripheral mantle of mononuclear cells composed of macrophages, epithelioid cells and lymphocytes; these histological changes were associated with a bacterial dissemination limited to the superficial lymph nodes. A lymphoid hyperplasia with prominent germinal centers was observed in the draining lymph nodes from day 3 PI. These results illustrate the dual role of granulomatous lesions in chronic bacterial infections: although they limit bacterial dissemination, the granulomas do not impair the persistence of infectious organisms in the host, leading to focal tissue damage.

Experimental Corynebacterium pseudotuberculosis infection in lambs: kinetics of bacterial dissemination and inflammation.

Infection and pyogranulomas induced by Corynebacterium pseudotuberculosis were experimentally reproduced in lambs. In two separate experiments, bacterial multiplication and dissemination were studied in 30 male lambs inoculated subcutaneously into the right ear with 1.1 or 1.5 X 10(8) viable C. pseudotuberculosis strain 19R. Infected lambs were necropsied at various times until the 28th day following inoculation. After a transient hyperthermia and a strong local inflammatory reaction, an abscess developed in the right ear from postinoculation day (PID) 6; it enlarged until PID 14 and stabilized thereafter and was associated with adenopathy of lymph nodes draining the head. Three acute phase indicators of inflammation were followed in 14 out of 30 lambs; plasma levels of copper and haptoglobin increased rapidly following inoculation whereas zinc levels decreased. The peaks were reached from PID 1 to 5, and thereafter the values came back slowly to the baseline. Antibodies against C. pseudotuberculosis exotoxin increased from PID 5 and reached a plateau on PID 21. Bacterial dissemination, assessed by the number of infected organs per lamb, was maximal on PID 16 and then stabilized until the end of the experiment. Lungs were infected in seven out of 18 lambs necropsied on PID 28. These results demonstrate a significant relationship between the clinical score of superficial lymph nodes or inoculation site and the infection level of these organs, and an early localization of pyogranulomatous lesions in regional lymph nodes. The subsequent development of the disease was related to the enlargement of these lesions and, in some animals, to a bacterial dissemination from primary sites of infection in the right prescapular lymph node and in the lung.

Antimicrobial edible packaging based on cellulosic ethers, fatty acids, and nisin incorporation to inhibit Listeria innocua and Staphylococcus aureus.

Edible cellulosic films made with hydroxypropylmethylcellulose (HPMC) have proven to be inadequate moisture barriers. To improve its water vapor barrier properties, different hydrophobic compounds were incorporated into the HPMC matrix. Some fatty acids and derivatives were included into the film-forming solution prior to film formation. Stearic acid was chosen because of its high capacity to reduce significantly the water vapor transmission rate. Antimicrobial activity of edible HPMC film was obtained by the incorporation of nisin into the film-forming solution. Nisin is an antimicrobial peptide effective against gram-positive bacteria. The inhibitory activity of this bacteriocin was tested for inhibition of Listeria innocua and Staphylococcus aureus. The use of stearic acid was observed to reduce the inhibitory activity of active HPMC film against both selected strains. This phenomenon may be explained by electrostatic interactions between the cationic nisin and the anionic stearic acid. Further studies showed that antimicrobial activity of film varied with the nature of the hydrophobic compound incorporated, in decreasing order: film without lipid, methylstearate film, and stearic acid film. This corroborated the idea of electrostatic interactions.

Low persistence of a large-plasmid-cured variant of Salmonella enteritidis in ceca of chicks.

In order to estimate the contribution of Salmonella in the persistence of this bacterium in chicks, we compared the persistence of a Salmonella enteritidis strain and its plasmid-cured variant in a chicken asymptomatic carrier state model. After oral inoculation, colonization with the plasmid-cured strain was significantly reduced (P < 0.001) in the ceca of chicks from the third week postinoculation and persisted for a shorter period than the wild-type strain. Moreover, numbers of S. enteriditis-infected livers were also significantly lower (P < 0.01) for the plasmid-cured strain compared with the wild-type strain from the third to the seventh week postinoculation. No difference in spleen colonization was observed. These results did not correlate with any in vitro difference in attachment, entry to, or intracellular multiplication of bacteria within intestinal or macrophage avian cell lines.

Differences in frequency, level, and duration of cecal carriage between four outbred chicken lines infected orally with Salmonella enteritidis.

Four chicken lines, L2, B13, PA12 (egg-type), and Y11 (meat-type), were tested for experimental carrier state of Salmonella enteritidis (SE) in two identical trials. After oral inoculation of SE at 1 wk of age with 5 x 10(4) SE colony-forming units (CFU), 10 chickens per line were necropsied weekly for 6 wk and then every 8 or 15 days until the 12th week postinoculation (PI). Liver, spleen, ovary, and ceca were examined for level of SE colonization. Numbers of positive livers and spleens and levels of the challenge strain in these organs differed little between the four chicken lines. Only three positive ovaries were detected. According to the chicken line, ceca exhibited generally significant (P < 0.05) differences in the number of positive organs during weeks 5-11 PI, in the SE CFU levels (P < 0.05) in the first 5 wk PI and during weeks 8 and 10 PI, and in the duration of colonization. L2 and B13 chickens generally carried SE in their ceca at higher levels, in more animals, and for a longer time than PA12 and Y11 chickens. Y11 chickens were the most resistant to SE cecal colonization.

Quantification of experimental Salmonella enteritidis carrier state in B13 leghorn chicks.

Quantification of the carrier state of Salmonella enteritidis in chicks (i.e., persistent asymptomatic association of S. enteritidis with the host), should provide an optimized means for further investigations into this problem. We therefore developed an experimental carrier state model by oral inoculation of low doses (10(2)-10(4)) of S. enteritidis in B13 chicks at different ages. Liver, spleen, and ceca colonizations by the challenge strains were measured weekly by enumeration of S. enteritidis colony-forming units (CFU) for 7-12 weeks. High mortality rates, incompatible with the carrier state, were observed in chicks inoculated with 10(2) organisms of either a parental strain of S. enteritidis (5556) or a mutant resistant to streptomycin (Smr) and nalidixic acid (Nalr) (strain 1009) at 1 day old. Both strains colonized organs similarly, allowing us to use subsequently the SmrNalr mutant strain. The selected low doses of S. enteritidis induced no deaths in chicks inoculated at 1 or 3 weeks of age. However, inoculation of 3-week-old chicks did not induce a satisfactory carrier state; organ colonization by S. enteritidis was weak and transient, even after inoculation of 10(8) SE. In contrast, some birds infected at 1 week of age presented the challenge strain in the liver and spleen for 3 weeks after inoculation and in the ceca for 12 weeks postchallenge. Most of these birds were colonized by S. enteritidis in the liver and in the ceca for 3 weeks and 10 weeks, respectively, following inoculation. Generally, CFU levels were highest during the first week(s) after inoculation and then decreased progressively. Levels of S. enteritidis were lower in the liver and spleen than in the ceca. Oral inoculation of 1-week-old birds with 5 x 10(4) S. enteritidis provided the required model, allowing quantification of the carrier state of S. enteritidis in chicks.