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.

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.

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.

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.

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.

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.

Diagnostic value of C-reactive protein and transthyretin in bone infections of the lower limb.

In a prospective study, white and red blood cell counts, hematocrit, erythrocyte sedimentation rate (ESR), albumin, alpha-1 acid glycoprotein, C-reactive protein (CRP), and transthyretin (TTR) values were determined by serial measurements during 23 days in 80 patients with an open fracture of the lower limb. Postoperative reference profiles were defined in 74 patients without septic complications. In the six remaining patients, serum CRP and TTR concentrations were found efficient for the early diagnosis of postoperative infections: a CRP/TTR mass concentration ratio higher than 0.6 from the 8th day after surgery was sensitive (100%) and specific (93%). Variations of CRP and TTR concentrations often preceded the clinical diagnosis in patients with early infection. ESR was found unreliable with regard to postoperative infection because of its high dependence with respect to red blood cell count.

Closed locked intramedullary nailing. Its application to comminuted fractures of the femur.

For many fractures of the femoral shaft, closed intramedullary nailing will not control rotation or telescoping of the fragments. Locked intramedullary nailing combines closed nailing with the percutaneous insertion of screws that interlock the bone and nail. This method permits static locking that controls rotation and telescoping and subsequently conversion to dynamic locking when weight-bearing is started after approximately twelve weeks. By providing greater stability, this method extends the indications for intramedullary nailing to severely comminuted, oblique, and spiral fractures as well as to fractures complicated by loss of bone and fractures in the proximal and distal ends of the femoral shaft. Of fifty-two patients with forty-nine severely comminuted fractures of the femoral shaft and three fractures that were complicated by loss of bone, forty-seven patients had uneventful consolidation of the fracture, with a mean time of 4.5 months for the severely comminuted fractures and seven months for the fractures that had a loss of bone. At follow-up, all forty-seven patients had normal motion of the hip, and forty-five had normal motion of the knee. Of the remaining five patients, four had a non-union that eventually healed (three after a second locked nailing and one after a third) and one had a septic non-union that eventually healed after removal of the nail and screws, débridement, and immobilization with an external fixator. Based on this experience, we concluded that this form of treatment has many advantages. The risk of infection and non-union is low, the incidence and severity of malunion are reduced, the hospital stay is short, and early mobilization of the patient is possible.

Comparison of serological tests for detection of Mycoplasma gallisepticum antibodies in eggs and chicks hatched from experimentally infected hens.

Specific pathogen free hens and males were experimentally infected with Mycoplasma gallisepticum. Eggs were then collected, and a part was incubated and set for hatching. Mycoplasma cultures were performed on infected adults and antibodies to MG were analysed by use of slide agglutination (SA) test and commercial ELISA tests on adults and chicks sera and on yolks from non incubated eggs. Both ELISA tests could detect antibodies in yolks from non incubated eggs laid three weeks after infection. SA and the three ELISA tests revealed positive sera in chicks hatched from eggs laid as soon as one week after infection.

Development of a blocking enzyme-linked immunosorbent assay for detection of turkey antibodies to Mycoplasma meleagridis.

A blocking enzyme-linked immunosorbent assay (B-ELISA) was developed to detect antibodies to Mycoplasma meleagridis (MM) in turkey sera. This assay was based on two mouse monoclonal antibodies recognising all MM strains tested but none of seven avian mycoplasmal species tested. Furthermore, their binding to the Tween 20 antigen was inhibited by serum from MM-infected birds. The B-ELISA test format was optimized. The cut-off was determined using a set of sera from MM-free turkeys. This B-ELISA was then compared with a commercial indirect ELISA (I-ELISA). Specificities of the two ELISA tests were not significantly different (100 or 99%, respectively). The sensitivity of B-ELISA was significantly higher than the I-ELISA when I-ELISA suspicious results were considered as negative. Testing sera from experimentally MM-infected animals showed that serum plate agglutination (SPA) test detected positive birds before both ELISA methods. Samples were collected in MM-infected commercial flocks and analyzed by SPA, ELISAs, MM-PCR or culture. Results showed that the sensitivity of the B-ELISA appeared superior to the I-ELISA. Moreover, the ability to detect maternal antibodies makes it a useful tool for eradication or control of MM infections.

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.

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.

Utility of an internal control for evaluation of a Mycoplasma meleagridis PCR test.

Mycoplasma meleagridis, a turkey pathogen, can be detected by PCR directly from tracheal or genital swabs. However, up to 40% samples may contain inhibitory substances. A DNA fragment, that can be amplified with M. meleagridis primers and in the same cycling conditions, was constructed to use as an internal control (IC) to check for these inhibitors. This IC can easily be distinguished from the M. meleagridis amplicon after agarose gel electrophoresis since it is longer. Use of this IC in PCR amplifications revealed that more than 35% of turkey tracheal swabs and more than 45% of turkey cloacal swabs contained inhibitors. In most cases, dilution (1:100) of swab lysates allowed amplification of the internal control but DNA purification may be necessary to eliminate inhibitors (20% of tracheal swabs and 5% of cloacal swabs). Use of this internal control DNA allowed assessment of the efficiency of each individual reaction and ensured that the reaction was not inhibited by interfering substances.

Antigen heterogeneity and epitope variable expression in Mycoplasma meleagridis isolates.

Immunoblotting was used to check the antigenic profiling of 27 Mycoplasma meleagridis strains isolated in different countries. Hyperimmune polyclonal rabbit antiserum as well as monoclonal antibodies (MAbs) raised against M. meleagridis (MM) showed antigen heterogeneity among strains. Five anti-MM MAbs were selected for lack of reaction against heterologous avian mycoplasma. Three of these five Mabs did not cross-react with 63 mycoplasma strains from six species affecting turkeys other than M. meleagridis. The five Mabs used to analyse the epitopes of 30 M. meleagridis strains indicated that some epitopes were not expressed in all strains. Moreover, other epitopes were located on proteins which differed according to number or molecular mass from strain to strain. The five Mabs therefore, recognised variable surface proteins, among which two were amphiphilic membrane proteins. Three of the selected Mabs recognised 29 or 30 of the 30 tested strains. The in vitro expression of surface epitopes in M. meleagridis ATCC 25284 was investigated by colony immunobinding and allowed demonstration of a variable antigenic system.

Detection of Mycoplasma synoviae in poultry environment samples by culture and polymerase chain reaction.

Successful detection of Mycoplasma synoviae (MS) by culture and PCR from samples collected in the environment of experimentally infected chickens and turkeys, or under field conditions, is described. Results showed that in the experimental infection, 10/96 and 46/96 samples of food, drinking water, feathers, droppings or dust were positive by culture and Mycoplasma-PCR. In field conditions, the number of positive results for environmental samples were respectively 7/28 and 17/28. These observations highlight the high disseminating capacities of this mycoplasma and show the usefulness of the PCR method for epidemiological studies.

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.