Investigation on eggshell apex abnormality (EAA) syndrome in France: isolation of Mycoplasma synoviae is frequently associated with Mycoplasma pullorum.

BACKGROUND: Mycoplasma synoviae (MS) is known to cause Eggshell Apex Abnormality (EAA) syndrome characterized by an altered shell surface with increased translucency on the apex. However, no large-scale studies have been conducted to obtain prevalence data of EAA and MS isolates associated to this syndrome. This manuscript reports the results of two field studies performed in the French poultry industry (2015-2017): focusing mainly on investigation of presence and prevalence of EAA in different types of laying hen flocks (phase 1), and isolation of MS strains from EAA-infected flocks (phase 2). RESULTS: The first survey included 77 farms of commercial layers in three French egg-production regions, hosting 40 flocks in alternative systems (ALT) and 56 in furnished cages (FC). Seven flocks (4 FC and 3 ALT) presented EAA clinical signs, giving a prevalence of 7.3% in this studied sample. A second independent field study was conducted to identify MS by in vitro cultivation and PCR in samples from 28 flocks with clinical signs of EAA. Different types of biological specimens were collected in EAA-affected flocks and submitted to the laboratory. M. synoviae was detected in 25/28 flocks, from both production systems (5/5 ALT and 20/23 FC). Detection of MS was significantly higher in tracheal swabs (59%) than in cloacal (10.5%), albumen (3.6%) and egg yolk (1.1%) swabs. It is worth to mention that attempts to clone MS from positive samples were often hampered by the presence of another Mycoplasma species, which showed fast growing behaviour in the selective media used in this study (Frey Medium 4 and Frey Medium 4 supplemented with erythromycin). The use of MALDI-TOF mass spectrometry in combination with next-generation sequencing (NGS) results allowed the identification of this fast growing mycoplasma as Mycoplasma pullorum, which was detected in 14 of the 25 (56%) MS-positive flocks. CONCLUSIONS: These results confirmed the presence of the EAA syndrome in MS-positive flocks of layers in France, reared in different regions and in different production systems (ALT and FC). Studies need to be conducted to test whether M. pullorum may influence the expression of clinical signs of EAA in MS-infected layer farms.

A new multiplex real-time TaqMan® PCR for quantification of Mycoplasma hyopneumoniae, M. hyorhinis and M. flocculare: exploratory epidemiological investigations to research mycoplasmal association in enzootic pneumonia-like lesions in slaughtered pigs.

AIMS: A new multiplex qPCR, targeting Mycoplasma (M.) hyopneumoniae, M. hyorhinis and M. flocculare, was developed and the relationship between detection of those mycoplasma species and the extent of gross pneumonia-like lesions in slaughtered pigs lungs were investigated. METHODS AND RESULTS: The multiplex qPCR method targets the p102, p37 and fruA genes and has detection limits of 14, 146 and 16 genome equivalents µl-1 for M. hyopneumoniae, M. hyorhinis and M. flocculare, respectively. In all, 671 lungs were collected and analysed, among them 666 were scored for macroscopic pneumonia and categorized according to the extent of the lesions (no or minor lesions, moderate lesions and extensive lesions). According to results of multiplex qPCR, 59·5% were positive for M. hyopneumoniae, 3·4% for M. hyorhinis and 34·7% for M. flocculare, with on average, 3·1 × 107 , 9·7 × 106 and 5·7 × 106 genome equivalents of mycoplasma ml-1 , respectively. More results showed that no or minor lesions were associated with multiplex qPCR-negative results or qPCR-positive results for M. flocculare. Moderate to extensive lesions were positively correlated with qPCR-positive results for M. hyopneumoniae. Extensive lesions were associated with qPCR-positive results for at least two mycoplasma species (M. hyopneumoniae and M. hyorhinis). CONCLUSION: The findings also indicated that M. hyopneumoniae and M. hyorhinis significantly increased the odds for a lung to have macroscopic pneumonia. No relationship was found between the extent of lesions and the mycoplasma genome load. SIGNIFICANCE AND IMPACT OF THE STUDY: This new multiplex qPCR appears to be specific, sufficiently sensitive and repeatable. The validation of this method with field samples guarantees its use for field epidemiological investigations, particularly to gain more insight into the aetiology of the porcine respiratory disease complex.

Quantification of Pasteurella multocida in experimentally infected pigs using a real-time PCR assay.

The aim of the study was to quantify Pasteurella multocida in experimentally infected pigs using a new qPCR assay based on the sodA gene and validated with 35 P. multocida strains, including strains isolated from pigs with pneumonia, clinically healthy pigs (nasal cavities), and human infections. The specificity of the test was verified with a collection of 60 strains of bacterial species other than P. multocida. The estimated detection threshold was 10 genome equivalents per microliter. The amplification efficiency and value of the correlation coefficients were 95.5% (±3.5%) and 0.995 (±0.005), respectively. Analysis of P. multocida suspensions in Buffered Peptone Water Broth and of samples prepared from lungs experimentally spiked with P. multocida revealed detection thresholds of 1.4CFU/µl and 8.4CFU/µl, respectively. In live pigs, experimentally-infected, approximately 105, 107 and 108genomeequivalents/ml of P. multocida DNA was detected on Day 8 post-infection in the nasal cavities, tonsils and trachea samples, respectively. In dead pigs, approximatively 107genomeequivalents/ml of P. multocida DNA was detected in the lung tissue with pneumonia. The qPCR assay's diagnostic specificity and sensitivity were 100% and 96%, respectively. This new qPCR assay should be a very useful tool for controlling enzootic pneumonia and studying the dynamics of infections in pig herds.

Escherichia coli Probiotic Strain ED1a in Pigs Has a Limited Impact on the Gut Carriage of Extended-Spectrum-ß-Lactamase-Producing E. coli.

Four trials were conducted to evaluate the impact of Escherichia coli probiotic strain ED1a administration to pigs on the gut carriage or survival in manure of extended-spectrum-ß-lactamase-producing E. coli Groups of pigs were orally inoculated with strain E. coli M63 carrying the blaCTX-M-1 gene (n = 84) or used as a control (n = 26). In the first two trials, 24 of 40 E. coli M63-inoculated pigs were given E. coli ED1a orally for 6 days starting 8 days after oral inoculation. In the third trial, 10 E. coli M63-inoculated pigs were given either E. coli ED1a or probiotic E. coli Nissle 1917 for 5 days. In the fourth trial, E. coli ED1a was given to a sow and its 12 piglets, and these 12 piglets plus 12 piglets that had not received E. coli ED1a were then inoculated with E. coli M63. Fecal shedding of cefotaxime-resistant Enterobacteriaceae (CTX-RE) was studied by culture, and blaCTX-M-1 genes were quantified by PCR. The persistence of CTX-RE in manure samples from inoculated pigs or manure samples inoculated in vitro with E. coli M63 with or without probiotics was studied. The results showed that E. coli M63 and ED1a were good gut colonizers. The reduction in the level of fecal excretion of CTX-RE in E. coli ED1a-treated pigs compared to that in nontreated pigs was usually less than 1 log10 CFU and was mainly observed during the probiotic administration period. The results obtained with E. coli Nissle 1917 did not differ significantly from those obtained with E. coli ED1a. CTX-RE survival did not differ significantly in manure samples with or without probiotic treatment. In conclusion, under our experimental conditions, E. coli ED1a and E. coli Nissle 1917 could not durably prevent CTX-RE colonization of the pig gut.

Impact of two different colistin dosing strategies on healthy piglet fecal microbiota.

Colistin is often used in piglets but underdosing and overdosing are frequent. The impact of such administrations on fecal microbiota was studied. Piglets were given either underdoses of colistin by oral gavage for five days or overdoses by in-feed medication for 14days. The composition of fecal microbiota was studied by quantitative PCR, 16S rRNA sequencing, culture of Enterobacteriaceae, and quantification of short-chain fatty acids (SCFAs). The mean colistin concentrations during the treatment for underdosed and overdosed groups were 14.4µg/g and 64.9µg/g of feces respectively. Whatever the piglet and the sampling day, the two main phyla were Firmicutes and Bacteroidetes, The main families were Lactobacillaceae, Clostridiales, Lachnospiraceae and Ruminococcaceae. The main perturbation was the significant but transitory decrease in the Escherichia coli population during treatment, yet all the E. coli isolates were susceptible to colistin. Moreover, colistin did not affect the production of SCFAs. These results show that under- or overdoses of colistin do not result in any major disturbance of piglet fecal microbiota and rarely select for chromosomal resistance in the dominant E. coli population.

Virulence Genes in Expanded-Spectrum-Cephalosporin-Resistant and -Susceptible Escherichia coli Isolates from Treated and Untreated Chickens.

This study investigated antimicrobial resistance, screened for the presence of virulence genes involved in intestinal infections, and determined phylogenetic groups of Escherichia coli isolates from untreated poultry and poultry treated with ceftiofur, an expanded-spectrum cephalosporin. Results show that none of the 76 isolates appeared to be Shiga toxin-producing E. coli or enteropathogenic E. coli. All isolates were negative for the major virulence factors/toxins tested (ehxA, cdt, heat-stable enterotoxin [ST], and heat-labile enterotoxin [LT]). The few virulence genes harbored in isolates generally did not correlate with isolate antimicrobial resistance or treatment status. However, some of the virulence genes were significantly associated with certain phylogenetic groups.

Impact of ceftiofur injection on gut microbiota and Escherichia coli resistance in pigs.

Resistance to extended-spectrum cephalosporins (ESCs) is an important health concern. Here, we studied the impact of the administration of a long-acting form of ceftiofur on the pig gut microbiota and ESC resistance in Escherichia coli. Pigs were orally inoculated with an ESC-resistant E. coli M63 strain harboring a conjugative plasmid carrying a gene conferring resistance, bla CTX-M-1. On the same day, they were given or not a unique injection of ceftiofur. Fecal microbiota were studied using quantitative PCR analysis of the main bacterial groups and quantification of short-chain fatty acids. E. coli and ESC-resistant E. coli were determined by culture methods, and the ESC-resistant E. coli isolates were characterized. The copies of the bla CTX-M-1 gene were quantified. After ceftiofur injection, the main change in gut microbiota was the significant but transitory decrease in the E. coli population. Acetate and butyrate levels were significantly lower in the treated group. In all inoculated groups, E. coli M63 persisted in most pigs, and the bla CTX-M-1 gene was transferred to other E. coli. Culture and PCR results showed that the ceftiofur-treated group shed significantly more resistant strains 1 and 3 days after ESC injection. Thereafter, on most dates, there were no differences between the groups, but notably, one pig in the nontreated group regularly excreted very high numbers of ESC-resistant E. coli, probably leading to a higher contamination level in its pen. In conclusion, the use of ESCs, and also the presence of high-shedding animals, are important features in the spread of ESC resistance.

Effect of land application of manure from enrofloxacin-treated chickens on ciprofloxacin resistance of Enterobacteriaceae in soil.

A field plot experiment was carried out to evaluate the impact of spreading chicken manure containing enrofloxacin (ENR) and its metabolite ciprofloxacin (CIP), on the levels of CIP-resistant Enterobacteriaceae in soil. The manures from chickens treated with ENR and from untreated control chickens were applied on six plots. Total and CIP-resistant Enterobacteriaceae were counted on Violet Red Bile Glucose medium containing 0 to 16mg L(-1) of CIP. A total of 145 isolates were genotyped by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). The minimum inhibitory concentration (MIC) of CIP for the isolates of each ERIC-PCR profile was determined. The most frequently isolated Enterobacteriaceae included Escherichia coli, and to a lesser extent, Enterobacter and 5 other genera from environmental origin. The composition of the E. coli community differed between manure and manured soil suggesting that the E. coli genotypes determined by ERIC-PCR varied significantly in their ability to survive in soil. One of these genotypes, including both susceptible and resistant isolates, was detected up to 89 days after the manure was applied. Most of the E. coli isolated in soil amended with manure from treated chickens was resistant to CIP (with a MIC ranging between 2 and 32mg L(-1)). In contrast, despite the presence of ENR in soil at concentrations ranging from 13-518µg kg(-1), the environmental Enterobacteriaceae isolates had a CIP MIC≤0.064mg L(-1), except one isolate which had a MIC of 0.25mg L(-1), These results showed that spreading manure from ENR-treated chickens enabled CIP-resistant E. coli to persist for at least three months in the soil. However, neither the presence of fluoroquinolones, nor the persistence of CIP-resistant E. coli, increased the CIP-susceptibility of environmental Enterobacteriaceae.

Gamma nail in the treatment of closed trochanteric fractures. Results and indications of 121 cases.

The Gamma Nail is the latest advance in the treatment of trochanteric fractures based on intramedullary nailing principles during closed procedures. Its design is based on Küntscher's Y-nail and locking intramedullary (IM) nails. This paper reports the results from the first-ever series of 121 patients operated between 1988 and 1990. They were followed until bone union was achieved. The mean patient age was 75 years. Most patients were in poor general health and had unstable fractures. Anatomical preoperative reduction was achieved in 72% of cases. Fixation was good in 66% of cases and acceptable in 27% of cases. Intra-operative complications consisted of nine fractures without consequences. Of the treated patients, 83.4% resumed weight-bearing during the first week. There was one case of deep infection that resolved with treatment. The mortality rate was 12.3% at three months. We noted 7 alunions in varus, 3 in valgus, 2 in external rotation and 1 in internal rotation. There were no cases of non-union. In six cases, the screw had cut out of the femoral head. The drawbacks associated with surgical treatment methods for trochanteric fractures also apply to the Gamma nail. Nevertheless, one of its primary advantages is the possibility of using a closed procedure. When compared to Ender nailing, knee pain is absent and weight-bearing can be achieved in all patients, no matter the fracture type.

Methicillin-resistant Staphylococcus aureus ST398 contamination and transmission in pigs after a low dose inoculation.

AIMS: Methicillin-resistant Staphylococcus aureus (MRSA) ST398 has recently been described as a zoonotic agent. Its transmission between animals seems to be a pivotal factor in its emergence and dissemination. This experimental trial was performed to describe MRSA ST398 contamination and transmission in pigs after a low dose inoculation. METHODS AND RESULTS: Twelve specific pathogen-free (SPF) pigs were randomly divided between two separate pens. Three pigs in each pen received a nasal inoculation of 2 × 10(4) colony-forming units per animal, and three naïve pigs were left in contact with them. Every 2 days and at necropsy, different samples were screened for MRSA. It was detected in nasal swabs from five inoculated and three naïve contact pigs, as early as 1 day after inoculation. MRSA was also found in environmental wipes but never in faecal samples. At necropsy, MRSA was detected in the lymph nodes of two contact pigs and in the tonsils and lymph nodes of three inoculated pigs. Twelve other SPF pigs were included as negative control in a separate room. CONCLUSION: This experiment showed that inoculation of a low dose of MRSA ST398 could lead to the horizontal transmission of the bacterium between pigs, the contamination of mandibular lymph nodes and the contamination of the environment without faecal carriage. SIGNIFICANCE AND IMPACT OF THE STUDY: The minimal inoculated dose via nasal route to observe transmission of MRSA ST398 between pigs is equal or lower to 2 × 10(4) colony-forming units per animal, and faecal excretion seems not to be a necessary condition for horizontal transmission.

[The cost of antibiotic resistance: analysis and consequences]. / Coût biologique de la résistance aux antibiotiques : analyse et conséquences.

Antimicrobial resistance, either by mutation or acquisition of resistance determinants harbored by mobile genetic elements, may confer a biological cost for the bacteria. This biological cost can be evaluated by comparing the resistant mutant to the wild susceptible strain, in the absence of antibiotic selection. This fitness cost can affect the growth rate in vitro or the survival in the host or in the environment or the virulence capacity. Various studies have evidenced this cost, either in vitro or in vivo, in different analysis models. However, bacteria can evolve and adapt to reduce this cost, by compensatory mutations or fine regulation of resistance expression. This compensatory evolution allows resistant bacteria to persist even in the absence of antibiotic selection pressure.

Comparison of genetic profiles of Campylobacter strains isolated from poultry, pig and Campylobacter human infections in Brittany, France.

Five hundred eighty-two Campylobacter isolates (177 from humans, 319 from poultry and 86 from pig) collected in Brittany, France, in 2003 and 2004 were typed by pulsed-field gel electrophoresis. The number of human cases increased during the hot season, particularly for C. jejuni. Twelve genetic groups out of 27 contained human isolates collected over the two years. These groups had 21.3 and 17.0% of the isolates obtained in 2003 and 2004, respectively. In four cases, isolates from 2003 have the same Pulsed-field gel electrophoresis (PFGE) profile as isolates from 2004. Six PFGE profiles common to poultry and human isolates were identified. Poultry isolates were found in 47 clusters containing human isolates. Caeca from farms and slaughterhouses accounted for 66% of these isolates, with chicken legs obtained from supermarkets accounting for the other 34%. Pig isolates never clustered with poultry and human isolates. In conclusion, the analysis of the genetic profiles of Campylobacter resulting from human cases showed that there were few identical or genetically close isolates between the human cases declared in 2003 and those declared in 2004. This highlighted a great genetic diversity in the isolates and indicated that it should be difficult to bind the human infections with groups of Campylobacter isolates presenting particular genetic profiles. The Campylobacter isolates obtained from the two animal production systems had different genotypes, and isolates from pigs differed genetically from isolates obtained from humans. We found that 44.6% of human Campylobacter isolates were genetically related to genotypes found in poultry and a part of these campylobacteriosis are due to contact with poultry. This is not particularly surprising in Brittany, a farming area with many animal-rearing farms and slaughterhouses. This work highlights the implication of the poultry in the French human cases and that handling of poultry is also an important risk for Campylobacter infection in humans.

Evidence for natural horizontal transfer of tetO gene between Campylobacter jejuni strains in chickens.

AIMS: The transfer of tetO gene conferring resistance to tetracycline was studied between Campylobacter jejuni strains, in the digestive tract of chickens. METHODS AND RESULTS: In vitro conjugation experiments were first performed in order to select donor/recipient couples for further in vivo assay. Then, chickens were inoculated with a donor/recipient couple of C. jejuni strains displaying spontaneous in vitro tetracycline resistance gene transfer. The donor was a tetracycline-resistant ampicillin-susceptible strain, and the recipient was a tetracycline-susceptible ampicillin-resistant strain. Chicken droppings were streaked on antimicrobial selective media and bi-resistant Campylobacter isolates were further characterized according to their donor or recipient flaA gene RFLP profile. The acquisition of tetracycline-resistance gene by the recipient C. jejuni strain from the donor C. jejuni strain was confirmed by tetO PCR. CONCLUSIONS: The study showed that transfer of tetO gene occurs rapidly and without antimicrobial selection pressure between C. jejuni strains in the digestive tract of chickens. SIGNIFICANCE AND IMPACT OF THE STUDY: The rapid and spontaneous transfer of tetO gene may explain the high prevalence of tetracycline resistance in chicken Campylobacter strains.

Fluoroquinolone resistance in Mycoplasma gallisepticum: DNA gyrase as primary target of enrofloxacin and impact of mutations in topoisomerases on resistance level.

Resistant mutants of Mycoplasma gallisepticum were selected in vitro by passaging strains 10 times in increasing concentrations of enrofloxacin. The regions of gyrA/gyrB and parC/parE, encoding the quinolone resistance-determining regions (QRDRs) of DNA gyrase and DNA topoisomerase IV, respectively, of the mutants obtained during different passages were sequenced. Several mutations were found in the four fluoroquinolone targets. Substitution of Ser-83-->Arg in GyrA and Ser-80-->Leu or Trp in ParC QRDRs seem to have the greatest impact on resistance to fluoroquinolones. The results obtained also suggest that the preferential target of enrofloxacin in M. gallisepticum is DNA gyrase.

A reverse transcription-PCR assay to detect viable Mycoplasma synoviae in poultry environmental samples.

In order to study horizontal transmission of Mycoplasma synoviae an avian pathogen, a reverse transcription-polymerase chain reaction (RT-PCR) assay was developed to detect viable Mycoplasma in environment. The test was based on the RT-PCR of the 16S ribosomal RNA (rRNA) of Mycoplasma genus. Results showed that Mycoplasma 16S rRNA was stable up to 23 h after cell death. Therefore, the test allowed detection of viable or very recently (less than 23 h) dead mycoplasmas. M. synoviae survival in artificially contaminated water, food and soil and in the environment of M. synoviae experimentally infected turkeys was estimated by culture and RT-PCR. The RT-PCR method was then applied in a naturally infected laying hen farm showing problems of recurrent mycoplasmosis in the hens. Results confirmed the usefulness of RT-PCR in checking the efficiency of biosecurity measures and in improving cleaning and disinfection protocols.

Antimicrobial susceptibility of Streptococcus suis isolated from swine in France and from humans in different countries between 1996 and 2000.

The susceptibility of 135 Streptococcus suis strains isolated from pigs (n = 110) and from humans (n = 25) to 13 antimicrobial agents was studied by microdilution and disc diffusion methods using Mueller-Hinton Agar II (MH) supplemented with either defibrinated sheep blood (MHSB) or horse serum (MHHS). Results were similar for both methods used except for penicillin G whose zone diameters were reduced with MHSB compared with MHHS. When MH was supplemented with sheep blood, 39% of S. suis strains classified as penicillin susceptible by MHHS microdilution showed intermediate susceptibility. Nearly all strains were susceptible to penicillin G (except by disc diffusion in MHSB), amoxicillin, ceftiofur, florfenicol, gentamicin and bacitracin. The least active antimicrobial agents were doxycycline and macrolides/lincosamides. High-level resistance (MIC > 500 mg/L or zone diameters < 10 mm) to streptomycin and kanamycin was detected in only a few strains. The virulence of strains did not seem to be related to antimicrobial resistance because no statistical difference was reported between the proportion of resistant strains of S. suis isolated from pigs with meningitis, septicaemia and arthritis, and those from tonsils and nasal cavities. However, significant differences were found in the proportions of macrolide- or doxycycline-resistant strains between S. suis serotype 2 and other serotypes. The results of antibiotic susceptibility testing presented in this study indicate that beta-lactams can be used in empirical treatment of human and pig S. suis infections in France.

In vitro development of resistance to enrofloxacin, erythromycin, tylosin, tiamulin and oxytetracycline in Mycoplasma gallisepticum, Mycoplasma iowae and Mycoplasma synoviae.

The in vitro emergence of resistance to enrofloxacin, erythromycin, tylosin, tiamulin, and oxytetracycline in three avian Mycoplasma species, Mycoplasma gallisepticum, Mycoplasma synoviae and Mycoplasma iowae was studied. Mutants were selected stepwise and their MICs were determined after 10 passages in subinhibitory concentrations of antibiotic. High-level resistance to erythromycin and tylosin developed within 2-6 passages in the three Mycoplasma species. Resistance to enrofloxacin developed more gradually. No resistance to tiamulin or oxytetracycline could be evidenced in M. gallisepticum or M. synoviae after 10 passages whereas, resistant mutants were obtained with M. iowae. Cross-sensitivity tests performed on mutants demonstrated that mycoplasmas made resistant to tylosin were also resistant to erythromycin, whereas mutants made resistant to erythromycin were not always resistant to tylosin. Some M. iowae tiamulin-resistant mutants were also resistant to both macrolide antibiotics. Enrofloxacin and oxytetracycline did not induce any cross-resistance to the other antibiotics tested. These results show that Mycoplasma resistance to macrolides can be quickly selected in vitro, and thus, providing that similar results could be obtained under field conditions, that development of resistance to these antibiotics in vivo might also be a relatively frequent event.

Characterization of mutations in DNA gyrase and topoisomerase IV Involved in quinolone resistance of Mycoplasma gallisepticum mutants obtained in vitro.

Mycoplasma gallisepticum enrofloxacin-resistant mutants were generated by stepwise selection in increasing concentrations of enrofloxacin. Alterations were found in the quinolone resistance-determining regions of the four target genes encoding DNA gyrase and topoisomerase IV from these mutants. This is the first description of such mutations in an animal mycoplasma species.

Comparison of pulsed-field gel electrophoresis with random amplified polymorphic DNA for typing of Mycoplasma synoviae.

Pulsed-field gel electrophoresis (PFGE) analysis was developed and compared with random amplified polymorphic DNA (RAPD) method to type 18 Mycoplasma synoviae (MS) strains. All analysed strains were typeable by RAPD but only 89% of MS strains were typeable by PFGE because of DNA degradation. The discriminatory power of RAPD was greater than that of PFGE but the two techniques had a discriminatory index superior to 0.95, the threshold value for interpreting typing results with confidence. The in vitro, in ovo and in vivo reproducibility of both typing techniques was 100%. However, the interpretation of RAPD patterns was complicated because of inconsistent band intensity. Thus, these molecular typing techniques should be helpful for epidemiological studies of avian mycoplasma infections.