Effects of antimicrobial exposure on the antimicrobial resistance of Escherichia coli in the digestive flora of dairy calves.

Veal calves are often identified as reservoirs for antimicrobial resistant Escherichia coli (E. coli). This production is closely linked with dairy production, as young calves - mostly males - are collected from dairy farms to enter the fattening process. The aim of this prospective study was to explore the factors on dairy farms that favour the selection of antimicrobial resistance (AMR) in the digestive E. coli strains of young calves and to assess whether the resistance levels and selection pressure were the same for males and females. The exposure of calves to antimicrobials was investigated through three factors: antimicrobial treatment of calves; feeding of calves with milk from cows treated with antimicrobials; and the consumption of colostrum from cows treated with antimicrobials at dry-off. The study design involved 100 dairy farms. A calf of each sex was selected from birth on each farm. Information on the calves' exposure to antimicrobials was collected daily and calves were sampled (rectal swab) two weeks after birth, then seven weeks after birth for females only. Laboratory analyses included culture on two distinct media: a non-selective medium (identifying dominant flora) and a medium containing ceftiofur to select the extended-spectrum beta-lactamase (ESBL) phenotype. Susceptibility testing was performed on an E. coli strain from each medium. Generalised linear models were used to assess associations between the resistance of E. coli strains and antimicrobial exposure. A set of 280 swabs from healthy calves were analysed. In dominant flora, high levels of resistance (>60 %) were identified for streptomycin, tetracycline and amoxicillin but AMR levels were low (3 %) for critically important antimicrobials (3rd- and 4th-generation cephalosporins and fluoroquinolones). For females staying in dairy farms, a marked decrease in resistance was observed for almost all antimicrobials between the age of 15 days and 7 weeks. A selective medium revealed an ESBL phenotype for 20.7 % of the calves. Whether for AMR or antimicrobial exposure, no significant difference was found between male and female calves. The antimicrobial treatment of calves was associated with an increased resistance of E. coli from dominant flora for amoxicillin (OR = 2.9), gentamicin (OR = 4.6), florfenicol (OR = 5.0) and trimethoprim-sulfonamide (OR = 5.6). The consumption by calves of milk from cows treated with antimicrobials was also associated with an increased resistance to amoxicillin (OR = 2.6), gentamicin (OR = 4.0), tetracycline (2.6) and trimethoprim-sulfonamide (OR = 2.2). In contrast, the models did not reveal any association between AMR and consumption of colostrum from cows treated with antimicrobials at dry-off.

Rural veterinarian's perception and practices in terms of biosecurity across three European countries.

The implementation of biosecurity measures in the animal health and production context is quite broad and aims at limiting the risk of introduction and spread of diseases. Veterinarians play a major role in biosecurity as key informants on the subject for cattle holders, key players in terms of disease prevention/control and eradication programs, as well as key risk factor in terms of disease dissemination. Many biosecurity studies have highlighted professional visitors such as veterinary practitioners as representing a high-risk factor in terms of disease introduction in animal facilities but, to date, very few studies have focused on the implementation level of biosecurity measures by veterinarians. An online survey was implemented in three European countries (Belgium, France and Spain) to assess the behaviour of rural veterinarians towards biosecurity, as well as their implementation level of the biosecurity measures. A descriptive analysis of data and a scoring system were applied to assess the implementation level of measures. The influence of different factors on the implementation level of biosecurity measures was investigated through a negative binomial regression model. The study identified different strengths, weaknesses, possible constraints and solutions in terms of veterinary perspectives. Veterinarians are considered as key informants by the farmers and could therefore play a more active role in terms of guidance and improvement of biosecurity at farm level. Based on the survey outcomes, two factors seemed to influence significantly the implementation level of measures: the country where he/she practices and the veterinarian's perception level of biosecurity. The biosecurity stages with the lowest application level, therefore representing the biggest threats, were bio-exclusion (increasing the risk of disease introduction) and biocontainment (increasing the risk of inter-herd transmission).

Immunogenicity of two Echinococcus granulosus antigens EgA31 and EgTrp in mice.

Here, we investigate in mice the immunogenicity of two antigens EgA31 and EgTrp which are expressed by the larval stage of Echinococcus granulosus. These recombinant proteins were used alone or as a mixture (EgA31-EgTrp) to immunize BALB/c mice. By flow cytometry, we have shown that the ratio CD4+/CD8+ of splenocytes were significantly higher in the antigen-immunized groups. The specific antibody in the sera and cytokine producing splenocytes was evaluated by enzyme-linked immunosorbent assay. EgA31, EgTrp or EgA31-EgTrp elicited high antibody titer of IgG and IgA. Among IgG isotypes, IgG1 was predominant for each antigen tested alone or combined. The production of IL-12, IFN-gamma, IL-10 and IL-6 cytokines was significantly higher in mice immunized with recombinant proteins. Our results suggest that, in BALB/c mice, a mixed Th1/Th2, response to EgA31, EgTrp and EgA31-EgTrp is obtained. The use of both antigens separately or in combination as candidate vaccine proteins is discussed.

An in vitro model to evaluate the cytokine response in Echinococcus infections.

With the aim of proposing an alternative model to animal experimentation, we investigated cytokine production in response to antigens in an in vitro system. This is a co-culture system of healthy human leukocytes and enterocyte-like Caco-2 cells. The antigens tested, EgA31, EgTrp, and FABP1, are candidates for vaccines in infections caused by Echinococcus spp. in the gut. All three have previously been described in the protoscolex stage and belong to protein families which confer protective immunity against several helminths. In this study, we evaluate the Th1/Th2 profile (Th1: IL-12, IFN-gamma; Th2: IL-6, IL-10) in response to protoscoleces, EgA31 and the mixture of EgA31, EgTrp and FABP1. No cytokine production was detected in response to protoscoleces. Neither IFN-gamma nor IL-6, but a significant IL-10 and IL-12 concentration was detected in response to both types of antigens. These findings suggest that EgA31 and the mixture EgA31/EgTrp/FABP1 generated an immunogenic response associated with a mixed Th1/Th2 cytokine.

Cellular immune response of lymph nodes from dogs following the intradermal injection of a recombinant antigen corresponding to a 66 kDa protein of Echinococcus granulosus.

A recombinant polypeptide (referred to as EgA31), which represents a 66kDa protein, was prepared from an Echinococcus granulosus cDNA library. In order to assess its potential to induce cellular immune responses, dog popliteal and prescapular lymph nodes were sensitized with this recombinant polypeptide. Subpopulations of lymphocytes were then analyzed by flow cytometry and immunohistochemistry on lymph node sections. Five days after the sensitization, the paracortical areas of the lymph nodes appeared hypertrophic, the number of CD3+, CD4+, CD8+ and CD5+ cells increased, the number of B-cells began to augment and some secondary follicles occurred, and a number of CD4+ cells appeared in germinal centers. Many large secondary follicles and a significantly augmented number of CD5+ cells in cords of medullae were observed 10 days after the sensitization. These active cellular responses strengthen the interest for further studies on the development of a vaccine with this recombinant polypeptide.

A new potent antigen from Echinococcus granulosus associated with muscles and tegument.

An immunoscreening of a cDNA library derived from the adult stage of the parasitic platyhelminth Echinococcus granulosus has been carried out with sera from infected dogs. The EgA31 clone encodes a fibrous protein which shares some sequence elements with paramyosins. The corresponding gene is present as a single copy in the genome. As revealed by an antibody obtained against a fusion protein produced in bacteria, the polypeptide has a molecular weight of 66 kDa. This polypeptide is present at all developmental stages studied and is a potent antigen during an infection by the adult stage in the dog and during cyst growth in human patients. By immunohistology, it was shown that it is present in the tegument and subtegumental parenchyma of the adult with a main location in the region of the suckers where it rapidly accumulates at the time of the head evagination. It is also present in the germinal layer of the cyst and on the protoscolex.

Identification and developmental expression of a Bombyx mori alpha-tubulin gene.

A cDNA clone isolated from the wing discs at the metamorphosis of Bombyx mori during the period of morphogenesis has been characterized. The amino acid sequence predicted for the putative protein is highly homologous to the Drosophila alpha1-tubulin. This is the first alpha-tubulin gene isolated in Bombyx mori and other isotype sequences are present in the Bombyx genome. The transcript is detected in the wing discs at every postembryonic stage examined, and is also expressed in other tissues, but at different levels. Although the mRNA level is maximum when the 20-hydroxyecdysone titre is high, its accumulation is independent of the hormone level both in vivo and in vitro. Significance of the accumulation of the mRNA of an ubiquitous alpha-tubulin in developing wing discs is discussed by comparison with our knowledge of the alpha-tubulin family in Drosophila and in other organisms.

Identification of a Bombyx collagenous protein with multiple short domains of Gly-Xaa-Yaa repeats: cDNA characterization and regulation of expression.

Using a differential screening strategy, a 2 kb cDNA was isolated from a differentiating Bombyx mori imaginal wing discs library. The available reading frame of 1710 bp specifies a conceptual protein with two distinct regions: a 395 amino acid region corresponding to 105 repetitions of the Gly-Xaa-Yaa triple-helical forming unit arranged in 9 domains of 12-54 residues interspaced by short interruptions of 3-17 residues and a C-terminal domain of 175 amino acids devoided of triplets. These characteristics suggest that the encoded protein is a new member of the collagen superfamily, the first one characterized in Bombyx. Northern blots reveal a unique 7.4 kb mRNA in imaginal wing discs, epidermis and brain. In wing discs, the 7.4 kb transcripts accumulate during the last larval moult and the spinning stage. In vitro studies reveal that this accumulation is dose dependent and detectable within 1 h in response to 20-hydroxyecdysone which acts without de novo protein synthesis. A model of regulation of transcript accumulation with regards to the hormonal level is proposed.

Expression of a Bombyx cytoplasmic actin gene in cultured Drosophila cells: influence of 20-hydroxyecdysone and interference with expression of endogenous cytoplasmic actin genes.

The expression of the Bombyx cytoplasmic actin A3 gene and its response to 20-hydroxyecdysone are studied after transfection in hormone responsive Drosophila cells and are compared to the expression of homologous resident genes. The host cells accumulate correct transcripts of the Bombyx gene in a gene dosage dependent way. The relative amount of endogenous cytoplasmic actin mRNAs is decreased in transfected cells, whether the transgene is integrated into the genome or not. When 20-hydroxyecdysone is added to the culture medium, the accumulation of the foreign mRNA is decreased whereas those of endogenous cytoplasmic actin transcripts are increased. These results are discussed in terms of competition for transcription and regulatory factors.

Genes expressed during the differentiation of the pupal wings are also transiently expressed during the larval moult in Bombyx mori.

By a differential screening of cDNA libraries made with RNAs present in differentiating imaginal wing discs of Bombyx mori, we have isolated clones whose expression is dramatically modified during the formation of the pupal wings. The RNAs corresponding to all but one of these clones are also transiently accumulated in the wing primordia during the last larvo-larval moult and in response to 20-hydroxyecdysone injections. If the injections are made to juvenilized larvae the expression of the same genes is stimulated but the range of the stimulation is reduced. It is suggested that in Bombyx, cells of the wings reach the pupal stage of differentiation by steps corresponding to the phases during which high levels of ecdysteroids are present.

[Effects of starvation on incorporation of glycine in organs of Philosamia cynthia walkeri]. / Effets du jeûne sur l'incorporation in vivo de glycine dans les organes de Philosamia cynthia walkeri.

The influence of starvation on protein synthesis in organs from growing Philosamia larvae has been determined. In the fat body, a rapid and continuous decrease in synthesis begins at the onset of starvation. A decrease also occurs in the carcass, but of lesser extent. In the intestine and silkgland, the synthesis increases significantly during the first 12 hours and decreases thereafter. In all organs, the acid soluble glycine pools are drastically modified by starvation for 48 hours: this amino-acid accumulates in the silkglands, remains at a rather stable level in the intestine but decreases in the fat body and carcass. No adjustement of these pools is found at the level of haemolymph acid soluble glycine which increases during the 48 hours. These results are discussed in the context of regulatory mechanisms on metabolic level during feeding or food deprivation periods.

Occurrence of an active regulatory mechanism of protein synthesis during starvation and refeeding in Bombyx mori fat body.

The origin of the amino acids which participate in protein synthesis at the recovery from starvation have been determined in the fat body from Bombyx mori larvae. Endogeneous amino acids have been labelled with [3H] leucine and ingested ones with [14C] leucine, allowing their discrimination in the organism. 22 minutes after refeeding, proteosynthetic activity of the fat body, estimated by the polysome level, is increased 2.5 fold. Endogeneous leucine represents more than 90 p. cent of the leucine present in nascent polypeptides. Free leucine pools of the fat body and of hemolymph increase, mainly through the release of endogeneous leucine. It is therefore concluded that refeeding with amino acids induces the production of a signal or critical factor, responsible for the increase in proteosynthetic activity in the fat body.

INFLUENCE DU JEÛNE AU NIVEAU ORGANIQUE CHEZ PHILOSAMIA CYNTHIA WALKERI. POIDS ET INCORPORATION A LONG TERME D'ACIDES AMINÉS.

The weight of organs and course of long term incorporation of amino acids into proteins are followed during a 48 hr starvation in Philosamia cynthia walkeri. In each organ, both parameters follow synchroneous evolutions: decrease of weight and amount of radio-active proteins since the beginning of starvation in the fat body; rapid increase during the first 6-12 hr in the intestine and integument, then a decrease. In hemolymph, the amount of radio-active proteins increases during 6hr and is stable thereafter. In the same time, the acid-soluble radio-activity is more depressed in the fat body than in hemolymph, and less in the integument and silkgland than in hemolymph. These results are discussed in relation with the influence of the fat body on growth and metabolism regulations during starvation. Two main hypothesis are suggested: intense exportation of amino acids from fat body to hemolymph in order to maintain a high level of material in the internal milieu; continuation of the growth of organs during the first hrs of starvation partly supported by the fat body.