Comparison of the Clinical Manifestation of HPAI H5Nx in Different Poultry Types in the Netherlands, 2014-2022.

This study describes clinical manifestations of highly pathogenic avian influenza (HPAI) H5N1, H5N8 and H5N6 outbreaks between 2014 and 2018 and 2020 and 2022 in the Netherlands for different poultry types and age groups. Adult duck (breeder) farms and juvenile chicken (broiler and laying pullet) farms were not diagnosed before 2020. Outbreaks in ducks decreased in 2020-2022 vs. 2014-2018, but increased for meat-type poultry. Neurological, locomotor and reproductive tract signs were often observed in ducks, whereas laying- and meat-type poultry more often showed mucosal membrane and skin signs, including cyanosis and hemorrhagic conjunctiva. Juveniles (chickens and ducks) showed neurological and locomotor signs more often than adults. Diarrhea occurred more often in adult chickens and juvenile ducks. Mortality increased exponentially within four days before notification in chickens and ducks, with a more fluctuating trend in ducks and meat-type poultry than in layers. For ducks, a mortality ratio (MR) > 3, compared to the average mortality of the previous week, was reached less often than in chickens. A lower percentage of laying flocks with MR > 3 was found for 2020-2022 vs. 2014-2018, but without significant differences in clinical signs. This study provides a basis for improvements in mortality- and clinical-sign-based early warning criteria, especially for juvenile chickens and ducks.

Recommendation of a standardized broth microdilution method for antimicrobial susceptibility testing of Avibacterium paragallinarum and resistance monitoring.

This study aimed to develop a method for standardized broth microdilution antimicrobial susceptibility testing (AST) of Avibacterium (Av.) paragallinarum, the causative agent of infectious coryza in chickens. For this, a total of 83 Av. paragallinarum isolates and strains were collected from 15 countries. To select unrelated isolates for method validation steps, macrorestriction analyses were performed with 15 Av. paragallinarum. The visible growth of Av. paragallinarum was examined in six broth media and growth curves were compiled. In Veterinary Fastidious Medium and cation-adjusted Mueller-Hinton broth (CAMHB) + 1% chicken serum + 0.0025% NADH (CAMHB + CS + NADH), visible growth of all isolates was detected and both media allowed adequate bacterial growth. Due to the better readability of Av. paragallinarum growth in microtiter plates, CAMHB + CS + NADH was chosen for AST. Repetitions of MIC testing with five epidemiologically unrelated isolates using a panel of 24 antimicrobial agents resulted in high essential MIC agreements of 96%-100% after 48-h incubation at 35 ± 2°C. Hence, the remaining 78 Av. paragallinarum were tested and demonstrated easily readable MICs with the proposed method. Differences in MICs were detected between isolates from different continents, with isolates from Africa showing lower MICs compared to isolates from America and Europe, which more often showed elevated MICs of aminoglycosides, quinolones, tetracyclines, and/or trimethoprim/sulfamethoxazole. PCR analyses of isolates used for method development revealed that isolates with elevated MICs of tetracyclines harbored the tetracycline resistance gene tet(B) but none of the other tested resistance genes were detected. Therefore, whole-genome sequencing data from 62 Av. paragallinarum were analyzed and revealed the presence of sequences showing nucleotide sequence identity to the genes aph(6)-Id, aph(3″)-Ib, blaTEM-1B, catA2, sul2, tet(B), tet(H), and mcr-like. Overall, the proposed method using CAMHB + CS + NADH for susceptibility testing with 48-h incubation time at 35 ± 2°C in ambient air was shown to be suitable for Av. paragallinarum. Due to a variety of resistance genes detected, the development of clinical breakpoints is highly recommended. IMPORTANCE: Avibacterium paragallinarum is an important pathogen in veterinary medicine that causes infectious coryza in chickens. Since antibiotics are often used for treatment and resistance of the pathogen is known, targeted therapy should be given after resistance testing of the pathogen. Unfortunately, there is currently no accepted method in standards that allows susceptibility testing of this fastidious pathogen. Therefore, we have worked out a method that allows harmonized susceptibility testing of the pathogen. The method meets the requirements of the CLSI and could be used by diagnostic laboratories.

Colistin Use in European Livestock: Veterinary Field Data on Trends and Perspectives for Further Reduction.

Polymyxin E (colistin) is a medically important active substance both in human and veterinary medicine. Colistin has been used in veterinary medicine since the 1950s. Due to the discovery of the plasmid-borne mcr gene in 2015 and the simultaneously increased importance in human medicine as a last-resort antibiotic, the use of colistin for animals was scrutinised. Though veterinary colistin sales dropped by 76.5% between 2011 to 2020, few studies evaluated real-world data on the use patterns of colistin in different European countries and sectors. A survey among veterinarians revealed that 51.9% did not use or ceased colistin, 33.4% decreased their use, 10.4% stabilised their use, and 2.7% increased use. The most important indications for colistin use were gastrointestinal diseases in pigs followed by septicaemia in poultry. A total of 106 (16.0%) responding veterinarians reported governmental/industry restrictions regarding colistin use, most commonly mentioning "use only after susceptibility testing" (57%). In brief, colistin was perceived as an essential last-resort antibiotic in veterinary medicine for E. coli infections in pigs and poultry, where there is no alternative legal, safe, and efficacious antimicrobial available. To further reduce the need for colistin, synergistic preventive measures, including improved biosecurity, husbandry, and vaccinations, must be employed.

Antimicrobial resistance monitoring in commensal and clinical Escherichia coli from broiler chickens: Differences and similarities.

BACKGROUND: In the Netherlands, antimicrobial resistance (AMR) is monitored in commensal indicator Escherichia coli from healthy broilers at slaughter as part of a European monitoring programme. In a separate programme for poultry health, AMR is monitored in veterinary pathogens from diseased broilers. So far, it is unknown how the outcomes of these two AMR monitoring approaches in the same animal population are associated. AIMS: This study aims to investigate the association between the outcomes of monitoring non-wildtype susceptibility (using epidemiological cut-off values, ECOFF, as prescribed by EU legislation) in commensal E. coli isolated from healthy broilers (i.e. active surveillance) with the outcomes of monitoring clinical resistance (using clinical breakpoints, to determine susceptibility for antibiotic treatment in veterinary practice) in E. coli isolated from diseased broilers (i.e. passive surveillance). METHODS: Data acquired by broth microdilution was analysed for commensal indicator E. coli and clinical E. coli from the Netherlands, 2014-2019. A generalized linear multivariable model (Poisson regression) was used to determine time trends and identify differences in mean resistant proportions. RESULTS: Observed resistant proportions of the monitored commensal E. coli and clinical E. coli were similar with overlapping confidence intervals for most time points for ampicillin, gentamicin, cefotaxime, tetracycline, colistin and trimethoprim/sulfonamide. The statistical analysis showed that only for cefotaxime and tetracycline, mean resistant proportions were different. In commensal E. coli, a decrease of resistant proportions over time was observed, except for gentamicin. In clinical E. coli, no time trend was detected in resistant proportions, except for cefotaxime and colistin. CONCLUSIONS: Generally, the resistant proportions monitored in commensal and clinical E. coli were similar. However, some relevant differences were found, which can be explained by the type of monitoring approach, i.e. active or passive surveillance. The random sample of commensal E. coli isolated from healthy animals (active surveillance), was more suitable to monitor AMR time trends. The sample of clinical isolates from diseased animals (passive surveillance), resulted in a higher chance to detect low-prevalent resistance: i.e. cefotaxime and colistin. The clinical E. coli data showed more fluctuation over time, and data from a longer period of time would be needed to determine the association. This study shows the value of both an active and a passive surveillance component for AMR monitoring.

Seasonal risk of low pathogenic avian influenza virus introductions into free-range layer farms in the Netherlands.

Poultry can become infected with avian influenza viruses (AIV) via (in) direct contact with infected wild birds. Free-range chicken farms in the Netherlands were shown to have a higher risk for introduction of low pathogenic avian influenza (LPAI) virus than indoor chicken farms. Therefore, during outbreaks of highly pathogenic avian influenza (HPAI), free-range layers are confined indoors as a risk mitigation measure. In this study, we characterized the seasonal patterns of AIV introductions into free-range layer farms, to determine the high-risk period. Data from the LPAI serological surveillance programme for the period 2013-2016 were used to first estimate the time of virus introduction into affected farms and then assess seasonal patterns in the risk of introduction. Time of introduction was estimated by fitting a mathematical model to seroprevalence data collected longitudinally from infected farms. For the period 2015-2016, longitudinal follow-up included monthly collections of eggs for serological testing from a cohort of 261 farms. Information on the time of introduction was then used to estimate the monthly incidence and seasonality by fitting harmonic and Poisson regression models. A significant yearly seasonal risk of introduction that lasted around 4 months (November to February) was identified with the highest risk observed in January. The risk for introduction of LPAI viruses in this period was on average four times significantly higher than the period of low risk around the summer months. Although the data for HPAI infections were limited in the period 2014-2018, a similar risk period for introduction of HPAI viruses was observed. The results of this study can be used to optimize risk-based surveillance and inform decisions on timing and duration of indoor confinement when HPAI viruses are known to circulate in the wild bird population.

Mutations potentially associated with decreased susceptibility to fluoroquinolones, macrolides and lincomycin in Mycoplasma synoviae.

Mycoplasma synoviae is one of the economically most significant avian Mycoplasma species. It can cause great financial losses to the poultry industry by inducing respiratory diseases, infectious synovitis, or eggshell apex abnormalities. There are different approaches to control M. synoviae infection. Although antimicrobial therapy cannot replace long-term solutions, like eradication and vaccination, this strategy can be effective in the short term, as adequate antibiotic treatment can relieve economic losses through the attenuation of clinical signs and reduction of transmission. Using broth microdilution method, minimal inhibitory concentration (MIC) values to fourteen antibiotics related to eight antimicrobial groups were determined in 96 M. synoviae strains. Whole genome sequencing and sequence analysis revealed mutations potentially associated with decreased susceptibility to fluoroquinolones, macrolides and lincomycin. Molecular markers responsible for the high MICs to fluoroquinolones were found in the gyrA, gyrB, parC and parE genes. Besides, single nucleotide polymorphisms identified in genes encoding the 23S rRNA were found to be responsible for high MICs to the 50S inhibitor macrolides and lincomycin, while amino acid change in the 50S ribosomal protein L22 could be associated with decreased susceptibility to macrolides. The revealed mutations can contribute to the extension of knowledge about the genetic background of antibiotic resistance in M. synoviae. Moreover, the explored potentially resistance-related mutations may serve as targets for molecular biological assays providing data of antibiotic susceptibility prior to the laborious and time-consuming isolation of M. synoviae strains.

Rate of false positive reactions in 11 M. gallisepticum and M. synoviae serological tests in samples obtained from SPF birds inoculated with heterologous mycoplasma species.

No recent information is available on the specificity of current M. synoviae (Ms) and M. gallisepticum (Mg) serological tests. In this study the performance of a currently available Mg and Ms Rapid Plate Agglutination (RPA) test, and three Mg, three Ms and three Mg/Ms combination ELISAs were evaluated on SPF sera that were obtained from days (D) 0-28 after M. gallinarum, M. imitans or M. gallinaceum inoculation, after sham inoculation and without inoculation. Tracheal swabs for mycoplasma culture were obtained before inoculation (D0), 7 and 28 days post inoculation (d.p.i.) in all groups except the sham inoculated group. The different mycoplasma species colonized well. In the early stage after inoculation (7-14 d.p.i.) with heterologous mycoplasma species, the specificity varied from 85% to 100% in the Mg RPA test and from 70% to 85% in the Ms RPA test. The specificity of both Mg and Ms RPA test was 100% in the sham inoculated samples and ruled out the effect of sham medium. In the late stage (21-28 d.p.i.) specificity was 100% for both RPA tests. The test specificity was 100% for seven ELISAs except for two combination ELISAs where a specificity of 95% was found in the late stage after inoculation. However, this was not significantly different from the specificity of all other tests in the late stage of these groups. These results show that it is not advisable to establish Mg and Ms seromonitoring programmes on the Mg and Ms RPA test alone as other mycoplasma species frequently occur in poultry.

Antimicrobial susceptibility of Avibacterium paragallinarum isolates from outbreaks of infectious coryza in Dutch commercial poultry flocks, 2008-2017.

The objective of the present study was to determine the in vitro antimicrobial susceptibility of Avibacterium paragallinarum isolates from infectious coryza outbreaks in Dutch commercial poultry, from 2008 till mid-2017. By using a broth microdilution method, minimal inhibitory concentrations (MICs) of 15 antimicrobial agents were assessed, and MIC50 and MIC90 values were determined. Additionally, isolates were subjected to different PCRs for the presence of genes that may confer antimicrobial resistance. Besides field isolates, a set of reference strains, among which the nine Kume strains and one Page serovar strain, were included in the study. For broth microdilution testing a new growth medium, recently developed for susceptibility testing of Haemophilus parasuis, was used. The medium proved to be suitable for broth microdilution susceptibility testing of NAD dependent Av. paragallinarum as well; visible growth was obtained in growth control wells and accepting a deviation of one dilution step, MIC values were reproducible. Results of 44 field isolates originating from 25 outbreaks showed relatively good susceptibility to antimicrobial agents that are recommended for the treatment of infectious coryza in the Netherlands, except for tetracycline; circa 75% of the isolates were characterized by MIC values of tetracycline of ≥16 µg/ml. In almost a quarter of these isolates with high MICs of tetracycline, tet genes were detected. For the remaining isolates with elevated MIC values, the mechanism conferring resistance remains to be studied. Of most agents, low MIC values were determined for the nine Kume and one Page serovar reference strains, as well as negative PCR results for resistance genes, being concordant with agar diffusion results reported for these strains.