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.

A Salmonella Pullorum outbreak with neurological signs in adult layers and outbreak investigation using whole genome sequencing.

RESEARCH HIGHLIGHTS: Cerebral granulomas are associated with nervous signs in Salmonella Pullorum outbreak.Bone marrow is also a recommended tissue for isolation of Salmonella Pullorum.Rapid plate agglutination test detects Pullorum antibodies in a vaccinated flock.Phylogenetic analysis showed clonality of isolates within the outbreak.

Mycoplasma bradburyae sp. nov. isolated from the trachea of sea birds.

In the search for mollicutes in wild birds, six Mycoplasma strains were isolated from tracheal swabs taken from four different species of seabirds. Four strains originated from three Yellow-legged gulls (Larus michahellis) and a Cory's shearwater (Calonectris borealis) from Spain, one from a South African Kelp gull (Larus dominicanus), and one from an Italian Black-headed gull (Chroicocephalus ridibundus). These Mycoplasma strains presented 99 % 16S rRNA gene sequence similarity values with Mycoplasma (M.) gallisepticum. Phylogenetic analyses of marker genes (16S rRNA gene and rpoB) confirmed the close relationship of the strains to M. gallisepticum and M. tullyi. The seabirds' strains grew well in modified Hayflick medium, and colonies showed typical fried egg morphology. They produced acid from glucose and mannose but did not hydrolyze arginine or urea. Transmission electron microscopy revealed a cell morphology characteristic of mycoplasmas, presenting spherical to flask-shaped cells with an attachment organelle. Gliding motility was also observed. Furthermore, serological tests, MALDI-ToF mass spectrometry and genomic studies demonstrated that the strains were different to any known Mycoplasma species, for which the name Mycoplasma bradburyae sp. nov. is proposed; the type strain is T158T (DSM 110708 = NCTC 14398).

Molecular characterization of the HMTp210 gene of Avibacterium paragallinarum and the proposition of a new genotyping method as alternative for classical serotyping.

Avibacterium paragallinarum (A. paragallinarum) is the aetiological agent of infectious coryza (IC) in chickens and characterized by acute respiratory distress and severe drop in egg production. Vaccination is important in the control of IC outbreaks and the efficacy of vaccination is dependent on A. paragallinarum serovars included in the vaccine. Classical serotyping of A. paragallinarum is laborious and hampered by poor availability of antigens and antisera. The haemagglutinin, important in classical serotyping, is encoded by the HMTp210 gene. HMTp210 gene analysis has been shown to have potential as alternative to classical serotyping. The aim of the present study was to further investigate the potential of sequence analyses of partial region 1 of the HMTp210 gene, the HMTp210 hypervariable region and the concatenated sequences of both fragments. For this analysis, 123 HMTp210 gene sequences (field isolates, A. paragallinarum serovar reference strains and vaccine strains) were included. Evaluation of serovar references and vaccine strains revealed a need for critical evaluation, especially within Page serovar B and C. Phylogenetic analysis of HMTp210 region 1 resulted in a separation of Page serovar A, B and C strains. Analysis of the HMTp210 HVR alone was not sufficient to discriminate all nine different Kume serovar references. The concatenated sequences of HMTp210 region 1 and HMTp210 HVR resulted in 14 clusters with a high correlation with Page serovar and with the nine currently known Kume serovars and is therefore proposed as a novel genotyping method that could be used as an alternative for classical serotyping of A. paragallinarum.

Genomic Diversity of a Globally Used, Live Attenuated Mycoplasma Vaccine.

The Mycoplasma synoviae live attenuated vaccine strain MS-H (Vaxsafe MS; Bioproperties Pty., Ltd., Australia) is commonly used around the world to prevent chronic infections caused by M. synoviae in birds and to minimize economic losses in the poultry industry. MS-H is a temperature-sensitive strain that is generated via the chemical mutagenesis of a virulent M. synoviae isolate, 86079/7NS. 32 single nucleotide polymorphisms have been found in the genome of MS-H compared to that of 86079/7NS, including 25 in predicted coding sequences (CDSs). There is limited information on the stability of these mutations in MS-H in vitro during the propagation of the vaccine manufacturing process or in vivo after the vaccination of chickens. Here, we performed a comparative analysis of MS-H genomes after in vitro and in vivo passages under different circumstances. Studying the dynamics of the MS-H population can provide insights into the factors that potentially affect the health of vaccinated birds. The genomes of 11 in vitro laboratory passages and 138 MS-H bird reisolates contained a total of 254 sequence variations. Of these, 39 variations associated with CDSs were detected in more than one genome (range = 2 to 62, median = 2.5), suggesting that these sequences are particularly prone to mutations. From the 25 CDSs containing previously characterized variations between MS-H and 86079/7NS, 7 were identified in the MS-H reisolates and progenies examined here. In conclusion, the MS-H genome contains individual regions that are prone to mutations that enable the restoration of the genotype or the phenotype of wild-type 86079/7NS in those regions. However, accumulated mutations in these regions are rare. IMPORTANCE Preventative measures, such as vaccination, are commonly used for the control of mycoplasmal infections in poultry. A live attenuated vaccine strain (Vaxsafe MS; MS-H; Bioproperties Pty. Ltd., Australia) is used for the prevention of disease caused by M. synoviae in many countries. However, information on the stability of previously characterized mutations in the MS-H genome is limited. In this study, we performed a comparative analysis of the whole-genome sequences of MS-H seeds used for vaccine manufacturing, commercial batches of the vaccine, cultures minimally passaged under small-scale laboratory and large-scale manufacturing conditions, MS-H reisolated from specific-pathogen-free (SPF) chickens that were vaccinated under controlled conditions, and MS-H reisolated from vaccinated commercial poultry flocks around the world. This study provides a comprehensive assessment of genome stability in MS-H after in vitro and in vivo passages under different circumstances and suggests that most of the mutations in the attenuated MS-H vaccine strain are stable.

Clinical expression, epidemiology, and monitoring of Mycoplasma gallisepticum and Mycoplasma synoviae: an update.

Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are of clinical and economic importance for the global poultry industry. Many countries and integrations are involved in monitoring programmes to control both mycoplasma species. This review provides an extensive historic overview of the last seven decades on the development of the knowledge regarding the factors that influence the clinical expression of the disease, the epidemiology, and monitoring of both MG and MS. This includes the detection of new virulent strains, studies unravelling the transmission routes, survival characteristics, and the role of other avian hosts. Also the role of molecular typing tests in unravelling epidemiology and factors that complicate the interpretation of test results is discussed. The latter includes the presence of heterologous mycoplasma infections, the use of heterologous oil-emulsion vaccines, and the use of antibiotic treatments. Also the occurrence of MG and MS strains with low virulence and the use of live and/or inactivated MS and MS vaccines are discussed.

High specificity of the Salmonella Pullorum/Gallinarum rapid plate agglutination test despite vaccinations against Salmonella Enteritidis and Salmonella Typhimurium.

In Europe, monitoring of breeding stock for Salmonella Pullorum (SP) or Salmonella Gallinarum (SG) infections is compulsory at the point of lay. Vaccinations against Salmonella Enteritidis (SE) and Salmonella Typhimurium (ST) are increasingly administered in Europe. These vaccines might induce cross-reactions in the rapid plate agglutination (RPA) SP/SG test due to shared O-antigens, possibly resulting in a lower test specificity. The extent to which the specificity of SP/SG serological tests is influenced by SE and/or ST vaccinations in the field has not been reported. In this paper, we report the diagnostic and flock specificity of the commercially available RPA SP/SG test using 1:2-1:16 serum dilutions on four panels of sera: SPF sera, field sera from flocks of varying age and SE/ST vaccination status, and reference sera from an international proficiency testing scheme. The results showed that the use of live SE/ST vaccines did not influence the specificity of the RPA SP/SG test. Inactivated vaccines showed a drop of the diagnostic specificity to 96.54% and a flock specificity of 34.1% when the 1:2 serum dilution was used. The 1:8 serum dilution showed a diagnostic specificity of 99.41% and a flock specificity of 86.4%. In conclusion, the use of SE/ST vaccines has either no effect or a modest effect on the specificity of the RPA SP/SG test used to monitor flocks. The main factors are the type of vaccine, and the serum dilution used for testing and a cut-off.

Identification, HPG2 Sequence Analysis, and Antimicrobial Susceptibility of Avibacterium paragallinarum Isolates Obtained from Outbreaks of Infectious Coryza in Commercial Layers in Sonora State, Mexico.

This is the first extensive report on the identification and characterization of Avibacterium paragallinarum (AVP) isolates obtained from outbreaks of infectious coryza (IC) in IC-vaccinated layer flocks from Sonora State in Mexico. Isolates obtained from IC outbreaks during the years 2007, 2014, 2015, 2017, and 2019 were identified by conventional PCR test and 16S rRNA gene analysis, serotyped by Page serotyping and genotyped by the recently described partial sequence analysis of the HPG2 region. Furthermore, antimicrobial susceptibility profiles were determined by a recently improved minimal inhibitory concentration (MIC) test. The conventional PCR test and the 16S rRNA analyses confirmed the isolates as AVP. Serotyping results showed the involvement of isolates belonging to serotypes A, B, and C in the IC outbreaks. Genotyping of the HPG2 region revealed the presence of sequence type (ST)1, ST4, and ST11, of which the latter has also been identified in Europe. The MIC susceptibility test showed that all tested isolates were susceptible for the majority of tested antimicrobials, including erythromycin and tetracycline, which are important antibiotics for the treatment of IC. The IC situation in Sonora State, Mexico, is complex because of the presence of serotypes A, B, and C. This finding emphasizes the importance of biosecurity in combination with the application of the most optimal vaccination programs in the control of IC in Sonora State, Mexico.

Nota de investigación­Análisis de secuencias de la región HPG2 y susceptibilidad antimicrobiana de aislamientos de Avibacterium paragallinarum obtenidos de brotes de coriza infecciosa en aves de postura comerciales en el estado de Sonora, México. Este es el primer informe extenso sobre la identificación y caracterización de aislamientos de Avibacterium paragallinarum (AVP) obtenidos de brotes de coriza infecciosa (IC) de parvadas de ponedoras vacunadas con coriza infecciosa en el estado de Sonora en México. Los aislamientos obtenidos de los brotes de coriza infecciosa durante los años 2007, 2014, 2015, 2017 y 2019 se identificaron mediante una prueba de PCR convencional y el análisis del gene de ARNr 16S, se serotipificaron mediante el método de Page y se genotipificaron mediante el análisis parcial de secuencias descrito recientemente de la región HPG2. Además, se determinaron los perfiles de susceptibilidad a los antimicrobianos mediante la prueba de concentración mínima inhibitoria (MIC) que ha sido mejorada recientemente. La prueba de PCR convencional y los análisis de secuencias del gene ARNr 16S confirmaron que los aislados eran A. paragallinarum. Los resultados de la serotipificación mostraron la participación de aislamientos pertenecientes a los serotipos A, B y C en los brotes de coriza infecciosa. La genotipificación de la región HPG2 reveló la presencia de secuencias del tipo (ST) 1, ST4 y ST11, de los cuales este último también ha sido identificada en Europa. La prueba de susceptibilidad por concentración mínima inhibitoria mostró que todos los aislados analizados eran susceptibles a la mayoría de los antimicrobianos analizados, incluida la eritromicina y la tetraciclina, que son antibióticos importantes para el tratamiento contra la coriza infecciosa. La situación de coriza infecciosa en el estado de Sonora, México, es compleja por la presencia de los serotipos A, B y C. Este hallazgo enfatiza la importancia de la bioseguridad en combinación con la aplicación de los programas de vacunación óptimos en el control de la coriza infecciosa en el estado de Sonora, México.

Development of molecular assays for the rapid and cost-effective determination of fluoroquinolone, macrolide and lincosamide susceptibility of Mycoplasma synoviae isolates.

Mycoplasma synoviae infection occurs worldwide, leading to considerable economic losses in the chicken and turkey industry due to infectious synovitis, respiratory diseases and eggshell apex abnormalities. Control programs against M. synoviae infection are based on eradication, vaccination and medication with antimicrobial agents. Prudent use of antibiotics can be improved greatly by the determination of antibiotic susceptibility prior to the treatment. However, the conventional broth or agar microdilution is very labor-intensive and time-consuming method. Thus, there is an increasing need for rapid antimicrobial susceptibility tests in order to guide antibiotic therapy more effectively. The aim of this study was to develop mismatch amplification mutation assays (MAMAs) to detect resistance-associated mutations in M. synoviae. M. synoviae strains with previously determined minimal inhibitory concentrations (MICs) and whole genomes (n = 92) were used for target selection and assay specification. For the evaluation of the developed assays, 20 clinical samples and an additional 20 M. synoviae isolates derived from these specimens were also included in this study. MIC values of these 20 isolates were determined by broth microdilution method. Five MAMAs were designed to identify elevated MICs of fluoroquinolones, while three MAMAs were developed to detect decreased susceptibility to macrolides and lincomycin. The sensitivity of the MAMA tests varied between 102-104 template copy number/reaction depending on the assay. Clinical samples showed identical genotype calls with the M. synoviae isolates derived from the corresponding specimens in each case. Supporting the results of conventional in vitro sensitivity tests, our approach provides a feasible tool for diagnostics. Rapidity, robustness and cost-effectiveness are powerful advantages of the developed assays. Supporting prudent antibiotic usage instead of empirical treatment, the use of this method can reduce significantly the economic impact of M. synoviae in the poultry industry and decrease bacterial resistance-related public health concerns.

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.

Limited changes in the fecal microbiome composition of laying hens after oral inoculation with wild duck feces.

Interspecies transmission of fecal microbiota can serve as an indicator for (indirect) contact between domestic and wild animals to assess risks of pathogen transmission, e.g., avian influenza. Here, we investigated whether oral inoculation of laying hens with feces of wild ducks (mallards, Anas platyrhynchos) resulted in a hen fecal microbiome that was detectably altered on community parameters or relative abundances of individual genera. To distinguish between effects of the duck inoculum and effects of the inoculation procedure, we compared the fecal microbiomes of adult laying hens resulting from 3 treatments: inoculation with wild duck feces (duck), inoculation with chicken feces (auto), and a negative control group with no treatment. We collected cloacal swabs from 7 hens per treatment before (day 0), and 2 and 7 D after inoculation, and performed 16S rRNA amplicon sequencing. No distinguishable effect of inoculation with duck feces on microbiome community (alpha and beta diversity) was found compared to auto or control treatments. At the individual taxonomic level, the relative abundance of the genus Alistipes (phylum Bacteroidetes) was significantly higher in the inoculated treatments (auto and duck) compared to the control 2 D after inoculation. Seven days after inoculation, the relative abundance of Alistipes had increased in the control and no effect was found anymore across treatments. These effects might be explained by the perturbation of the hen's microbiome caused by the inoculation procedure itself, or by intrinsic temporal variation in the hen's microbiome. This experiment shows that a single inoculation of fecal microbiota from duck feces to laying hens did not cause a measurable alteration of the gut microbiome community. Furthermore, the temporary change in relative abundance for Alistipes could not be attributed to the duck feces inoculation. These outcomes suggest that the fecal microbiome of adult laying hens may not be a useful indicator for detection of single oral exposure to wild duck feces.

Identification and characterization of Dutch Avibacterium paragallinarum isolates and the implications for diagnostics.

This study reports the results of diagnostic and molecular typing methods for 18 Avibacterium paragallinarum isolates obtained from outbreaks of infectious coryza in commercial layer flocks in the Netherlands. Isolation, biochemical identification, species-specific PCR tests and classical serotyping were performed. In addition, molecular typing by Enterobacterial Repetitive Intergenic Consensus-Based Polymerase Chain Reaction (ERIC-PCR) and sequence analysis of the partial HPG2 region of A. paragallinarum were applied and results of both techniques were compared. Moreover, the pathogenicity of an isolate of the most common genotype detected in the Netherlands was determined in an animal experiment. All 18 Avibacterium isolates were nicotinamide adenine dinucleotide-dependent. All isolates were detected by the species-specific conventional PCR while 33% of the isolates were missed by the species-specific real-time PCR. Sequence analysis showed a probe mismatch as a result of a single nucleotide polymorphism (G1516A). Modification of the probe of the real-time PCR was necessary to overcome false negative results. Molecular typing showed that sequence analysis of the partial HPG2 region was in concordance with ERIC-PCR results and indicated the presence of two major genotypes. Serotyping showed the presence of serovars A-1, A-2 and B-1. There was no correlation between genotyping results and serotyping results. Inoculation of an isolate of the most prevalent genotype, and belonging to serovar A-1, into brown layer hens demonstrated the pathogenicity of this isolate.

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.

Seroprevalence of Mycoplasma synoviae in Dutch commercial poultry farms.

Before the year 2000, Mycoplasma synoviae was associated mainly with subclinical respiratory infections in broilers in the Netherlands and was considered to have low clinical and economic impact. The subsequent occurrence of M. synoviae arthritis and amyloid arthropathy, and of eggshell apex abnormalities, has resulted in an increasing demand for M. synoviae-free poultry. Therefore, a cross-sectional seroprevalence study was carried out over a 12-month period during 2005 and 2006. Ten blood samples per farm were generally used because M. synoviae was expected to spread quickly. However, for grandparent and layer breeder stock, 24 to 60 blood samples per house were available from a voluntary M. synoviae monitoring programme. Sera were tested by means of the rapid plate agglutination test (agglutination at dilution > or =1:8 was considered positive). The numbers of farms sampled out of the national total were: broiler grandparent, 53/53; broiler parent rearing, 34/150; broiler parent, 114/300; broiler, 185/800; layer grandparent, 13/13; layer parent, 40/50; layer, 173/1250; and meat turkey, 50/75. The seroprevalence of M. synoviae in commercial poultry was high, especially in commercial layers where it was 73% (95% confidence interval (CI)=67 to 80); in layer and broiler grandparent stock, the seroprevalence was 0% and 10%, respectively, based on sample sizes equal to the population size. In layer and broiler parent farms, the seroprevalence was 25% (95% CI=19 to 31) and 35% (95% CI=28 to 44); in both broiler parent rearing and broiler farms it was 6% (95% CI=0 to 13 and 95% CI=3 to 9); and in meat turkey, the seroprevalence was 16% (95% CI=10 to 22).

Identification of Brachyspira hyodysenteriae and other pathogenic Brachyspira species in chickens from laying flocks with diarrhea or reduced production or both.

Cecal samples from laying chickens from 25 farms with a history of decreased egg production, diarrhea, and/or increased feed conversion ratios were examined for anaerobic intestinal spirochetes of the genus Brachyspira. Seventy-three samples positive in an immunofluorescence assay for Brachyspira species were further examined using selective anaerobic culture, followed by phenotypic analysis, species-specific PCRs (for Brachyspira hyodysenteriae, B. intermedia, and B. pilosicoli), and a Brachyspira genus-specific PCR with sequencing of the partial 16S rRNA gene products. Brachyspira cultures were obtained from all samples. Less than half of the isolates could be identified to the species level on the basis of their biochemical phenotypes, while all but four isolates (5.2%) were speciated by using PCR and sequencing of DNA extracted from the bacteria. Different Brachyspira spp. were found within a single flock and also in cultures from single chickens, emphasizing the need to obtain multiple samples when investigating outbreaks of avian intestinal spirochetosis. The most commonly detected spirochetes were the pathogenic species B. intermedia and B. pilosicoli. The presumed nonpathogenic species B. innocens, B. murdochii, and the proposed "B. pulli" also were identified. Pathogenic B. alvinipulli was present in two flocks, and this is the first confirmed report of B. alvinipulli in chickens outside the United States. Brachyspira hyodysenteriae, the agent of swine dysentery, also was identified in samples from three flocks. This is the first confirmed report of natural infection of chickens with B. hyodysenteriae. Experimental infection studies are required to assess the pathogenic potential of these B. hyodysenteriae isolates.

Genotyping of Mycoplasma gallisepticum and M. synoviae by Amplified Fragment Length Polymorphism (AFLP) analysis and digitalized Random Amplified Polymorphic DNA (RAPD) analysis.

Mycoplasma gallisepticum (MG) and M. synoviae (MS) are the cause of considerable economic losses in the poultry industry. Molecular differentiation of avian Mycoplasma strains may be helpful in tracing infections and in the evaluation of implemented intervention strategies. Amplified Fragment Length Polymorphism (AFLP) has shown to be a powerful typing technique but the application for poultry Mycoplasma strains is very limited. The aim of this study was to evaluate the reproducibility and discriminatory power of AFLP HindIII/HhaI and AFLP BglII/Mfel for the inter- and intraspecies differentiation of avian mycoplasmas and to compare these test characteristics with digitalized Random Amplified Polymorphic DNA (RAPD) analysis. The reproducibility of RAPD, AFLP HindIII/HhaI and AFLP BglII/Mfel was 50-100, 97-98 and 86-99%, respectively. RAPD and both AFLP enzyme combinations were able to differentiate between five avian Mycoplasma species. For AFLP, five MG and four MS clusters could be identified. The phylogenetic tree for both enzyme combinations was comparable. For RAPD, four MG clusters could be identified. For MS, however, due to the poor reproducibility of the RAPD technique, no clear genogroups could be identified. On basis of the results of this study it can be concluded that AFLP is a powerful technique for the genotyping of avian mycoplasmas and that, although AFLP HindIII/HhaI generated patterns with less fragments, the final results showed homologous results.