The aim of this study was to investigate the relationship between antimicrobial use and the occurrence of antimicrobial resistance in the digestive and respiratory tract in three different production systems of food producing animals. A longitudinal study was set up in 25 Belgian bovine herds (10 dairy, 10 beef, and 5 veal herds) for a 2 year monitoring of antimicrobial susceptibilities in E. coli and Pasteurellaceae retrieved from the rectum and the nasal cavity, respectively. During the first year of observation, the antimicrobial use was prospectively recorded on 15 of these farms (5 of each production type) and transformed into the treatment incidences according to the (animal) defined daily dose (TIADD) and (actually) used daily dose (TIUDD). Antimicrobial resistance rates of 4,174 E. coli (all herds) and 474 Pasteurellaceae (beef and veal herds only) isolates for 12 antimicrobial agents demonstrated large differences between intensively reared veal calves (abundant and inconstant) and more extensively reared dairy and beef cattle (sparse and relatively stable). Using linear mixed effect models, a strong relation was found between antimicrobial treatment incidences and resistance profiles of 1,639 E. coli strains (p<0.0001) and 309 Pasteurellaceae (p≤0.012). These results indicate that a high antimicrobial selection pressure, here found to be represented by low dosages of oral prophylactic and therapeutic group medication, converts not only the commensal microbiota from the digestive tract but also the opportunistic pathogenic bacteria in the respiratory tract into reservoirs of multi-resistance.

Mycoplasma gallisepticum is the most important pathogenic avian Mycoplasma species and causes chronic respiratory disease in poultry. In addition, the prevalence of Mycoplasma synoviae is of increasing concern in several EU member states. We investigated the prevalence of M. gallisepticum in commercial poultry (5220 layers, 1224 broilers and 1020 meat turkeys), 56 racing pigeons and 890 wild birds (Order Anseriformes, Galliformes, Pelecaniformes, Accipitriformes, Gruiformes, Charadriiformes, Columbiformes, Strigiformes, Falconiformes and Passeriformes). Broilers and wild birds were also evaluated for Mycoplasma synoviae. Dependent on the bird lifespan and the nature of the sample, different diagnostic tests were used including the rapid plate agglutination test, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction and real-time polymerase chain reaction. A low prevalence of M. gallisepticum was found in both layers (0.9%; 95% CI: 0.7-1.2%) and broilers (2.7%; 95% CI: 1.9-3.8%) possibly due to reduced vertical transmission by breeder farms, which are under official surveillance. None of the samples from turkeys or racing pigeons tested positive. In wild birds, we found five birds were positive (1.7%; 95% CI: 0.7-3.9%): one wood pigeon, two grey herons, one mallard and one Eurasian magpie. For M. synoviae a high prevalence was found in broilers (12.9%: 95% CI: 11.1-14.9%). Four samples collected by hunters gave a positive result for M. synoviae (4%: 95% CI: 1.6-9.8%): one carrion crow and three wood pigeons. In addition, 12 house sparrows were found to be positive (3%; 95% CI: 1.7-5.2%). Wild birds probably play a limited role as a reservoir but we cannot exclude a possible impact on transmission of Mycoplasmas.

19During the last few years, acquired resistance to colistin in , but also in other 20 bacterial species, has been reported. It disk diffusion assay, suggesting acquired resistance in 15 strains (9.6 %). The E-test and disk 29 prediffusion assay generated results within acceptable levels compared to the reference agar 30 dilution assay. The categorical agreement with the results obtained the agar dilution 31 method were good to very good for all tests, except the disk diffusion assay. In conclusion, 32 current results suggest that, in addition to the E-test, the disk prediffusion test is a reliable, 33 alternative agar-based colistin susceptibility method for testing colistin susceptibility of 34 isolates in diagnostic bacteriology.

To measure the level of antimicrobial resistance in potential bovine respiratory pathogens at different production types, nasal swabs were collected from 57 calves of 13 dairy herds, 150 calves of 9 beef cattle herds, and 289 calves of 5 high-density veal calf herds and investigated for the presence of Pasteurellaceae. All calves were less than 6 months old. Susceptibilities of the Pasteurella and Mannheimia isolates to eight antimicrobials were determined using an agar dilution method. P. multocida (37.3%) and hemolytic Mannheimia organisms (M. haemolytica sensu lato) (6.3%) were the most frequently detected organisms. The overall prevalence of isolates resistant to at least one antimicrobial from the dairy, beef, and veal calves were 17.6% (6/34), 21.9% (14/64), and 71.9% (64/89), respectively. In isolates obtained on the veal calf herds, acquired resistance to ampicillin, oxytetracycline, potentiated sulfonamides, gentamicin, tilmicosin, and enrofloxacin was frequently present, and 32.6% of these isolates were resistant to more than two of the tested antimicrobials. Resistance to ceftiofur and florfenicol was not detected. A substantial within-herd variability of species diversity and resistance profiles among isolates belonging to the genera Pasteurella and Mannheimia was found among the isolates of the veal calf farms.

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Virulence of Streptococcus ga//o/yticus (S. bovis) strains isolated from pigeons is associated with the presence of the extracellular proteins A, TI, T2 and T3. Based on the presence or absence of these proteins, six supernatantphenotypes are distinguished. Experimental infection studies have indicated that strains belonging to the A'TI, A+TI, A+T2 and A+T3 groups are highly virulent for pigeons, strains belonging to the A'T3 groups are moderately virulent and A'T2 strains are of low virulence. In this study the surface structure of 15 pigeon 5. ga//o/yticus strains representing high, moderate and low virulence supernatant-phenotypes was examined by electron microscopy. The presence of capsular material was determined by transmission electron microscopy after polycationic ferritin labelling and immunostabilization. Capsules from cells labelled with polycationic ferritin were usually thicker than those from cells exposed to antiserum. The capsule of the virulent strains had a regular, continuous appearance whilst irregularity of the capsule was a characteristic of the low virulence A'T2 strains. Negative staining revealed the presence of fimbriae in all strains belonging to the high virulence A'TI, A+TI, A+T2 and A+T3 supernatant groups and in one strain of the moderately virulent A'T3 group. The fimbriae were thin, flexible structures with a diameter of approximately 3-4 nm and a length of up to 700 nm. Fimbriae as described above were absent in two other A'T3 strains examined and in the low virulence A'T2 strains. Results from this study indicate that morphological differences in surface structure exist among virulent and low virulence pigeon S. ga//o/yticus strains, and that the capsule and/or fimbriae are possibly involved in virulence.

Cleaning and disinfection (C&D) of poultry houses is an essential aspect in farm hygiene management. Adequate performance of the different steps of a C&D protocol and the use of suitable products are key to prevent and control zoonoses and animal diseases. Hygiene monitoring on total aerobic flora through sampling with agar contact plates at different locations of the poultry house results in a hygienogram score that is used to evaluate the proper execution of C&D.This study analyzed the hygienogram scores of 19,739 poultry flocks in Flanders after C&D. Data relating to the C&D protocol, i.e., year, season, husbandry system, production type, cleaning product, sampler, active components of the disinfectant, disinfection time, disinfection temperature, and disinfection responsible, were collected.The average hygienogram score decreased significantly over time, suggesting a general improvement between 2007 and 2014. Differences in scores were found among the husbandry systems, with the barn/aviary system having a significantly better hygienogram score compared to the floor house, furnished cage, and battery. Significantly better scores also were found when a cleaning product was used in the C&D protocol. Disinfection with a peracetic acid and hydrogen peroxide combination or formaldehyde gave the best scores. In addition, C&D protocols using ≥2 different disinfectants showed improved results compared to the use of one single disinfectant. Finally, disinfection applied by a specialist contractor resulted in a better score compared to disinfection by the farmer.In conclusion, analysis of the hygienogram scores and related data allowed identifying several factors, resulting in an improvement, which may reduce the total bacterial load in poultry stables and, consequently, the number of zoonotic and pathogenic micro-organisms.