Actinobacillus pleuropneumoniae encodes multiple phase-variable DNA methyltransferases that control distinct phasevarions.

Actinobacillus pleuropneumoniae is the cause of porcine pleuropneumonia, a severe respiratory tract infection that is responsible for major economic losses to the swine industry. Many host-adapted bacterial pathogens encode systems known as phasevarions (phase-variable regulons). Phasevarions result from variable expression of cytoplasmic DNA methyltransferases. Variable expression results in genome-wide methylation differences within a bacterial population, leading to altered expression of multiple genes via epigenetic mechanisms. Our examination of a diverse population of A. pleuropneumoniae strains determined that Type I and Type III DNA methyltransferases with the hallmarks of phase variation were present in this species. We demonstrate that phase variation is occurring in these methyltransferases, and show associations between particular Type III methyltransferase alleles and serovar. Using Pacific BioSciences Single-Molecule, Real-Time (SMRT) sequencing and Oxford Nanopore sequencing, we demonstrate the presence of the first ever characterised phase-variable, cytosine-specific Type III DNA methyltransferase. Phase variation of distinct Type III DNA methyltransferase in A. pleuropneumoniae results in the regulation of distinct phasevarions, and in multiple phenotypic differences relevant to pathobiology. Our characterisation of these newly described phasevarions in A. pleuropneumoniae will aid in the selection of stably expressed antigens, and direct and inform development of a rationally designed subunit vaccine against this major veterinary pathogen.

The characterization and correlation between the phenotypic and genotypic resistance of Campylobacter spp. isolates from commercial broilers and native chickens in the south of Thailand.

RESEARCH HIGHLIGHTS: High Campylobacter prevalence in chickens; C. jejuni more prevalent than C. coli.Susceptibility to macrolides but resistance to quinolones/tetracyclines in isolates.Homogeneous resistance patterns within farms; higher in broilers than in native birds.Partial association between phenotypic and genotypic resistance among isolates.

Correction: Nucleic acid purification from plants, animals and microbes in under 30 seconds.

[This corrects the article DOI: 10.1371/journal.pbio.2003916.].

Molecular and serological characterization of Riemerella isolates associated with poultry in Australia.

A total of 62 isolates of Riemerella-like organisms, originally isolated from Australian poultry (10 from chickens, 46 from ducks, five from unknown hosts and one vaccine strain), were included in this study. On the basis of two published polymerase chain reaction (PCR) assays that are reported to be specific for Riemerella anatipestifer, 51 of the isolates were identified as R. anatipestifer. Forty-six of these isolates had a detailed history and were sourced from ducks, while five were of unknown origin. The 11 remaining isolates failed to yield a positive reaction in either PCR with 10 originating from chickens and one from a duck. Amplification and sequencing of the 16S rRNA gene of these isolates identified the duck isolate as Moraxella lacunta. Phylogenetic analysis of the 10 chicken isolates identified one as R. columbina and the remaining nine isolates as Riemerella-like taxon 2. The 51 Australian R. anatipestifer isolates were assigned by gel diffusion test to serovars 1 (26 isolates), 6 (seven isolates), 8 (five isolates), 9 (two isolates), 13 (one isolate) and 14 (one isolate) while nine isolates gave no reaction to any antiserum. A commercial system was used to perform DNA fingerprinting using rep-PCR analysis, which revealed different clusters with a lack of a clear relationship between the clusters and the serovars.

Detection of porcine circovirus type 2 (PCV2) in the Philippines and the complexity of PCV2-associated disease diagnosis.

Porcine circovirus type 2 (PCV2), an important pig viral pathogen, can cause porcine circovirus-associated disease (PCVAD), resulting in economic losses associated with decreased growth and mortalities. The diagnosis of PCVAD is complex requiring clinical, pathological and virological approaches. This study assessed PCV2 infection using histopathology and immunohistochemistry (IHC) on tissue samples and quantitative polymerase chain reaction (qPCR) on serum samples from 47 grower-finisher pigs allocated in three clinical groups in the Philippines. Typical PCV2 histopathological lesions were observed in mediastinal lymph nodes (MLN) of eight of 47 pigs. Lymphoid depletion was seen in all eight pigs and granulomatous inflammation in one of these pigs. Four of these eight pigs were PCV2 positive by both IHC and qPCR. IHC revealed PCV2 antigen in 8 pigs in at least one of the following tissues: MLN (5/8), spleen (3/8), tonsils (4/8) and lungs (5/8). PCV2 antigen was observed in 3/8 MLN with lymphoid depletion and in one MLN with depletion and granulomatous inflammation. The qPCR test showed that 33 sera had a non-detectable level, twelve had < 106 and two had > 106 PCV2 DNA copies/ml serum. One pig with lymphoid depletion had > 106 PCV2 DNA copies/ml serum, and another pig without MLN lesions also had > 106 PCV2 DNA copies/ml serum. These findings suggest that PCVAD is present in the Philippines and confirm the challenges of PCVAD diagnosis as different patterns of results were obtained from the different tests.

MOLECULAR IDENTIFICATION OF MEMBERS OF THE FAMILY PASTEURELLACEAE FROM THE ORAL CAVITY OF KOALAS (PHASCOLARCTOS CINEREUS) AND THEIR RELATIONSHIP WITH ISOLATES FROM KOALA BITE WOUNDS IN HUMANS.

A total of 22 Pasteurellaceae isolates obtained from the oral cavity of koalas (Phascolarctos cinereus) at different wildlife centers in Australia were investigated using amplification and sequencing of two housekeeping genes, rpoA and recN. The available sequences from the Lonepinella koalarum type strain (ACM3666T) and the recent isolates of Lonepinella-like bacteria obtained from human infected wounds associated with koala bites were also included. Phylogenetic analysis was performed on the concatenated rpoA-recN genes and genome relatedness was calculated based on the recN sequences. The oral cavity isolates, the koala bite wound isolates, and L. koalarum ACM3666T resulted in four clusters (Clusters 1-4). Clusters 1-3 were clearly not members of the genus Lonepinella. Cluster 1 was closely related to the genus Fredericksenia, and Clusters 2 and 3 appeared to be novel genera. Cluster 4 consisted of three subclusters: Cluster 4a with one koala bite wound isolate and L. koalarum ACM3666T, Cluster 4b with three oral cavity isolates and two Lonepinella-like wound isolates, and Cluster 4c with three nearly identical oral cavity isolates that may represent a different species within the genus Lonepinella. The rich Pasteurellaceae population, including potential novel taxa in the oral cavity of koalas supports an important role of these highly adapted microorganisms in the physiology of koalas. Moreover, the pathogenic potential of Lonepinella-like species is an important consideration when investigating infected koala bites in humans.

Nucleic acid purification from plants, animals and microbes in under 30 seconds.

Nucleic acid amplification is a powerful molecular biology tool, although its use outside the modern laboratory environment is limited due to the relatively cumbersome methods required to extract nucleic acids from biological samples. To address this issue, we investigated a variety of materials for their suitability for nucleic acid capture and purification. We report here that untreated cellulose-based paper can rapidly capture nucleic acids within seconds and retain them during a single washing step, while contaminants present in complex biological samples are quickly removed. Building on this knowledge, we have successfully created an equipment-free nucleic acid extraction dipstick methodology that can obtain amplification-ready DNA and RNA from plants, animals, and microbes from difficult biological samples such as blood and leaves from adult trees in less than 30 seconds. The simplicity and speed of this method as well as the low cost and availability of suitable materials (e.g., common paper towelling), means that nucleic acid extraction is now more accessible and affordable for researchers and the broader community. Furthermore, when combined with recent advancements in isothermal amplification and naked eye DNA visualization techniques, the dipstick extraction technology makes performing molecular diagnostic assays achievable in limited resource settings including university and high school classrooms, field-based environments, and developing countries.

A comparison of virulence genes, antimicrobial resistance profiles and genetic diversity of avian pathogenic Escherichia coli (APEC) isolates from broilers and broiler breeders in Thailand and Australia.

ABSTRACT Avian pathogenic Escherichia coli (APEC) is the causative agent of colibacillosis resulting in economic losses in the poultry industry worldwide. A total of 168 APEC isolates, equal numbers from Australian and Thai broilers/broiler breeders, were identified and tested for their susceptibility to ten antimicrobial agents. Most of the Thai APEC isolates were multidrug-resistant (MDR) (60.7%) whilst Australian APEC isolates showed a MDR rate of just 10.7%. The Thai APEC isolates exhibited high resistance to tetracycline (TET) (84.5%), amoxicillin (AMX) (70.2%) and trimethoprim-sulfamethoxazole (SXT) (51.2%) whilst the Australian APEC isolates showed lower levels of resistance (TET 36.9%, AMX 29.8%, SXT 17.86%). The 34 Thai APEC and four Australian APEC isolates which were resistant to nalidixic acid were characterized for their carriage of mutations in the quinolone resistance determining region of gyrA, gyrB, parC and parE. While no mutations were detected in gyrB in the Thai isolates, the Ser83Leu and Asp87Asn substitutions in gyrA and Ser80Ile in parC were common (n = 9/34). In regard to the Australian isolates, the Ser83Leu and Asp678Glu substitution in gyrA, Pro385Ala and Ser492Asn in gyrB and Met241Ile and Asp475Glu in parC were identified (n = 3/4). Rep-PCR analysis of the 84 Thai and 84 Australian APEC isolates showed 16 main clusters that mostly contained isolates from both countries. Our results suggest that the emergence of MDR is a major concern for the Thai APEC isolates and that more prudent use of antimicrobial agents in Thai poultry production is required.

Genotypic divergence of Avibacterium paragallinarum isolates with different growth requirements for nicotinamide adenine dinucleotide.

In 2017, for the first time in Asia, we reported the isolation of variants of Avibacterium paragallinarum with atypical NAD dependency. The present study was conducted to characterize the genotypes of 24 isolates of Av. paragallinarum in Korea, including the four variants reported previously. Most of the typical isolates (19/20) showed a unique ERIC-PCR pattern with no ERIC-PCR patterns in common between the typical isolates and the variants. Furthermore, the variants shared no ERIC-PCR patterns among themselves. All the typical NAD-dependent isolates belonged to the same phylogenetic group based on both 16S rRNA and hagA gene sequences. The four variants were placed in several groups distinct from the typical isolates. In the 16S rRNA phylogenetic analysis, two of the variants were not closely aligned to any other Av. paragallinarum, isolate although they were clearly members of the genus Avibacterium. The other variants were clustered together with NAD atypical isolates from geographically diverse global locations. Compared with the Modesto reference strain AY498870, all the variants lacked a TTTTT stretch at positions 182-186 in the 16S rRNA gene and the same deletion was shown in most of the reported variants. The typical isolates and variants shared 97.3-98.2% and 95.2-97.2% nucleotide sequence similarity, for 16S rRNA and hagA, respectively. In addition, the similarities among variants were within 98.3-100% and 96.5-98.4% for the two genes, respectively. Our results indicate that the Av. paragallinarum variants with altered NAD growth requirements were genetically different and highly divergent from the typical NAD-dependent isolates.RESEARCH HIGHLIGHTS NAD variant Korean Av. paragallinarum isolates show genetic diversity, whereas typical Korean Av. paragallinarum isolates do not.The Korean variants were not closely aligned to all other Av. paragallinarum in the 16S rRNA phylogeny.NAD atypical isolates from geographically diverse global locations clustered together.Almost all variants, including all Korean variants of Av. paragallinarum, lack a specific fragment of the 16S rRNA gene.

Streptococcus suis contains multiple phase-variable methyltransferases that show a discrete lineage distribution.

Streptococcus suis is a major pathogen of swine, responsible for a number of chronic and acute infections, and is also emerging as a major zoonotic pathogen, particularly in South-East Asia. Our study of a diverse population of S. suis shows that this organism contains both Type I and Type III phase-variable methyltransferases. In all previous examples, phase-variation of methyltransferases results in genome wide methylation differences, and results in differential regulation of multiple genes, a system known as the phasevarion (phase-variable regulon). We hypothesized that each variant in the Type I and Type III systems encoded a methyltransferase with a unique specificity, and could therefore control a distinct phasevarion, either by recombination-driven shuffling between different specificities (Type I) or by biphasic on-off switching via simple sequence repeats (Type III). Here, we present the identification of the target specificities for each Type III allelic variant from S. suis using single-molecule, real-time methylome analysis. We demonstrate phase-variation is occurring in both Type I and Type III methyltransferases, and show a distinct association between methyltransferase type and presence, and population clades. In addition, we show that the phase-variable Type I methyltransferase was likely acquired at the origin of a highly virulent zoonotic sub-population.

Towards a Standardized Method for Broth Microdilution Susceptibility Testing of Haemophilus parasuis.

Currently, there is no agreed method available for broth microdilution susceptibility testing of Haemophilus parasuis, one of the most important bacterial pathogens in pig production. Therefore, the aim of this study was to develop a method that could be easily performed by diagnostic laboratories and that appears suitable for a harmonized susceptibility testing. Growth determinations using one type strain and three field isolates revealed no visible growth of H. parasuis in media which have proven to be suitable for susceptibility testing of fastidious organisms. Therefore, a new medium, cation-adjusted Mueller-Hinton broth (CAMHB) plus NADH and sterile filtered heat-inactivated chicken serum, was developed. The reproducibility of MICs obtained in this medium was evaluated and statistically analyzed, considering a model with two different variables (precondition of five identical MICs and MIC mode accepting a deviation of ±1 dilution step, respectively). No significant differences for both variables were seen between two time points investigated and between results obtained with the recently proposed test medium broth (TMB). Nearly all MICs of quality control strains were in the acceptable range. Subsequently, 47 H. parasuis isolates representing 13 serovars were tested with the newly developed medium and TMB. Statistical analysis of all isolates and 15 antimicrobial agents and antimicrobial combinations showed no significant difference between MICs obtained in supplemented CAMHB and TMB. Because of a simplified implementation in routine diagnostic and a lower chance of interference between medium components and antimicrobial agents, supplemented CAMHB is recommended with an incubation time of 24 h.

Odour emissions from poultry litter - A review litter properties, odour formation and odorant emissions from porous materials.

Odour emissions from meat chicken sheds can at times cause odour impacts on surrounding communities. Litter is seen as the primary source of this odour. Formation and emission of odour from meat chicken litter during the grow-out period are influenced by various factors such as litter conditions, the environment, microbial activity, properties of the odorous gases and management practices. Odour emissions vary spatially and temporally. This variability has made it challenging to understand how specific litter conditions contribute to odour emissions from the litter and production sheds. Existing knowledge on odorants, odour formation mechanisms and emission processes that contribute to odour emissions from litter are reviewed. Litter moisture content and water thermodynamics (i.e. water activity, Aw) are also examined as factors that contribute to microbial odour formation, physical litter conditions and the exchange of individual odorant gases at the air-water interface. Substantial opportunities exist for future research on litter conditions and litter formation mechanisms and how these contribute to odour emissions. Closing this knowledge gap will improve management strategies that intercept and interfere with odour formation and emission processes leading to an overall reduction in the potential to cause community impacts.

Water activity of poultry litter: Relationship to moisture content during a grow-out.

Poultry grown on litter floors are in contact with their own waste products. The waste material needs to be carefully managed to reduce food safety risks and to provide conditions that are comfortable and safe for the birds. Water activity (Aw) is an important thermodynamic property that has been shown to be more closely related to microbial, chemical and physical properties of natural products than moisture content. In poultry litter, Aw is relevant for understanding microbial activity; litter handling and rheological properties; and relationships between in-shed relative humidity and litter moisture content. We measured the Aw of poultry litter collected throughout a meat chicken grow-out (from fresh pine shavings bedding material to day 52) and over a range of litter moisture content (10-60%). The Aw increased non-linearly from 0.71 to 1.0, and reached a value of 0.95 when litter moisture content was only 22-33%. Accumulation of manure during the grow-out reduced Aw for the same moisture content. These results are relevant for making decisions regarding litter re-use in multiple grow-outs as well as setting targets for litter moisture content to minimise odour, microbial risks and to ensure necessary litter physical conditions are maintained during a grow-out. Methods to predict Aw in poultry litter from moisture content are proposed.

Mapping the carriage of flaA-restriction fragment length polymorphism Campylobacter genotypes on poultry carcasses through the processing chain and comparison to clinical isolates.

Poultry are considered a major source for campylobacteriosis in humans. A total of 1866 Campylobacter spp. isolates collected through the poultry processing chain were typed using flaA-restriction fragment length polymorphism to measure the impact of processing on the genotypes present. Temporally related human clinical isolates (n = 497) were also typed. Isolates were obtained from whole chicken carcass rinses of chickens collected before scalding, after scalding, before immersion chilling, after immersion chilling and after packaging as well as from individual caecal samples. A total of 32 genotypes comprising at least four isolates each were recognised. Simpson's Index of Diversity (D) was calculated for each sampling site within each flock, for each flock as a whole and for the clinical isolates. From caecal collection to after packaging samples the D value did not change in two flocks, decreased in one flock and increased in the fourth flock. Dominant genotypes occurred in each flock but their constitutive percentages changed through processing. There were 23 overlapping genotypes between clinical and chicken isolates. The diversity of Campylobacter is flock dependant and may alter through processing. This study confirms that poultry are a source of campylobacteriosis in the Australian population although other sources may contribute.

Molecular identification and characterisation of Mannheimia haemolytica.

Mannheimia haemolytica is known as one of the major bacterial contributors to Bovine Respiratory Disease (BRD) syndrome. This study sought to establish a novel species-specific PCR to aid in identification of this key pathogen. As well, an existing multiplex PCR was used to determine the prevalence of serovars 1, 2 or 6 in Australia. Most of the 65 studied isolates originated from cattle with a total of 11 isolates from small ruminants. All problematic field isolates in the identification or serotyping PCRs were subjected to whole genome sequencing and bioinformatic analysis. The field isolates were also subjected to rep-PCR fingerprinting. A total of 59 out of the 65 tested isolates were conformed as M. haemolytica by the new species-specific PCR which is based on the rpoB gene. The confirmed M. haemolytica field isolates were assigned to serovars 1 (24 isolates), 2 (seven isolates) and 6 (26 isolates) while two of the isolates were negative in the serotyping PCR. The two non-typeable isolates were assigned to serovar 7 and 14 following whole genome sequencing and bioinformatic analysis. The rep-PCR typing resulted in five major clusters with serovars 1 and 6 often within the same cluster. The M. haemolytica-specific PCR developed in this work was species specific and should be a valuable support for frontline diagnostic laboratories. The serotyping results support the relative importance of serovars 1 and 6 in bovine respiratory disease.

Molecular characterization of Campylobacter spp. isolates obtained from commercial broilers and native chickens in Southern Thailand using whole genome sequencing.

Chickens are the primary reservoirs of Campylobacter spp., mainly C. jejuni and C. coli, that cause human bacterial gastrointestinal infections. However, genomic characteristics and antimicrobial resistance of Campylobacter spp. in low- to middle-income countries need more comprehensive exploration. This study aimed to characterize 21 C. jejuni and 5 C. coli isolates from commercial broilers and native chickens using whole genome sequencing and compare them to 28 reference Campylobacter sequences. Among the 26 isolates, 13 sequence types (ST) were identified in C. jejuni and 5 ST in C. coli. The prominent ST was ST 2274 (5 isolates, 19.2%), followed by ST 51, 460, 2409, and 6455 (2 isolates in each ST, 7.7%), while all remaining ST (464, 536, 595, 2083, 6736, 6964, 8096, 10437, 828, 872, 900, 8237, and 13540) had 1 isolate per ST (3.8%). Six types of antimicrobial resistance genes (ant(6)-Ia, aph(3')-III, blaOXA, cat, erm(B), and tet(O)) and one point mutations in the gyrA gene (Threonine-86-Isoleucine) and another in the rpsL gene (Lysine-43-Arginine) were detected. The blaOXA resistance gene was present in all isolates, the gyrA mutations was in 95.2% of C. jejuni and 80.0% of C. coli, and the tet(O) resistance gene in 76.2% of C. jejuni and 80.0% of C. coli. Additionally, 203 virulence-associated genes linked to 16 virulence factors were identified. In terms of phenotypic resistance, the C. jejuni isolates were all resistant to ciprofloxacin, enrofloxacin, and nalidixic acid, with lower levels of resistance to tetracycline (76.2%), tylosin (52.3%), erythromycin (23.8%), azithromycin (22.2%), and gentamicin (11.1%). Most C. coli isolates were resistant to all tested antimicrobials, while 1 C. coli was pan-susceptible except for tylosin. Single-nucleotide polymorphisms concordance varied widely, with differences of up to 13,375 single-nucleotide polymorphisms compared to the reference Campylobacter isolates, highlighting genetic divergence among comparative genomes. This study contributes to a deeper understanding of the molecular epidemiology of Campylobacter spp. in Thai chicken production systems.

Pasteurella multocida Heddleston serovar 3 and 4 strains share a common lipopolysaccharide biosynthesis locus but display both inter- and intrastrain lipopolysaccharide heterogeneity.

Pasteurella multocida is a Gram-negative multispecies pathogen and the causative agent of fowl cholera, a serious disease of poultry which can present in both acute and chronic forms. The major outer membrane component lipopolysaccharide (LPS) is both an important virulence factor and a major immunogen. Our previous studies determined the LPS structures expressed by different P. multocida strains and revealed that a number of strains belonging to different serovars contain the same LPS biosynthesis locus but express different LPS structures due to mutations within glycosyltransferase genes. In this study, we report the full LPS structure of the serovar 4 type strain, P1662, and reveal that it shares the same LPS outer core biosynthesis locus, L3, with the serovar 3 strains P1059 and Pm70. Using directed mutagenesis, the role of each glycosyltransferase gene in LPS outer core assembly was determined. LPS structural analysis of 23 Australian field isolates that contain the L3 locus revealed that at least six different LPS outer core structures can be produced as a result of mutations within the LPS glycosyltransferase genes. Moreover, some field isolates produce multiple but related LPS glycoforms simultaneously, and three LPS outer core structures are remarkably similar to the globo series of vertebrate glycosphingolipids. Our in-depth analysis showing the genetics and full range of P. multocida lipopolysaccharide structures will facilitate the improvement of typing systems and the prediction of the protective efficacy of vaccines.

Studies on the presence and persistence of Pasteurella multocida serovars and genotypes in fowl cholera outbreaks.

Pasteurella multocida was isolated from poultry on six farms (one free-range duck farm, one free-range turkey farm, one conventional enclosed turkey farm, and three free-range layer farms) suffering fowl cholera outbreaks. In addition, historical isolates from previous outbreaks were available for the conventional turkey farm and the three free-range layer farms. The isolates were serotyped using the Heddleston scheme and genotyped using multi-locus sequencing typing. In the current outbreaks, two of the farms had two different sequence types (STs) of P. multocida in the investigated outbreak (the free-range turkey farm and one of the free-range layer farms). The remaining four farms had one ST within the investigated outbreak. In looking at the historical isolates, two of the four farms had multiple genotypes involved. On the four farms with historical isolates from previous outbreaks, at least one new genotype was present in the investigated outbreak as compared with the historical isolates. On one layer farm, one genotype persisted over a 10-year period. Serotyping revealed the presence of multiple serovars in the current and historical outbreaks, with serovars sometimes changing over time. This study has shown that several STs of P. multocida can be present during some outbreaks of fowl cholera, although other outbreaks involve a single ST. Also, the STs present on a property suffering repeated fowl cholera can both persist and change over time.

LONEPINELLA SP. ISOLATED FROM WOUND INFECTIONS OF KOALAS.

We describe two cases of wound infections of koalas (Phascolarctos cinereus), one wild and one captive, in which Lonepinella-like organisms were involved. The wild adult koala was captured with bite wound injuries, as part of a koala population management program in Queensland, Australia. In both cases, there was evidence of physical trauma causing the initial wound. The captive koala suffered injury from the cage wire, and the wild koala had injuries suggestive of intermale fighting. Gram-negative bacteria isolated from both cases proved to be challenging to identify using routine diagnostic tests. The wound in the captive koala yielded a pure culture of an organism shown by whole genome sequence (WGS) analysis to be a member of the genus Lonepinella, but not a member of the only formally described species, L. koalarum. The wound of the wild koala yielded a mixed culture of Citrobacter koseri, Enterobacter cloacae and an organism shown by WGS analysis to be Lonepinella, but again not Lonepinella koalarum. Both cases were difficult to treat; the captive koala eventually had to have the phalanges amputated, and the wild koala required removal of the affected claw. The two Lonepinella isolates from these cases have a close relationship to an isolate from a human wound caused by a koala bite and may represent a novel species within the genus Lonepinella. Wound infections in koalas linked to Lonepinella have not been reported previously. Wildlife veterinarians need to be aware of the potential presence of Lonepinella-like organisms when dealing with wound infections in koalas, and the inability of commercial kits and systems to correctly identify the isolates.

Serovar identification, antimicrobial sensitivity, and virulence of Avibacterium paragallinarum isolated from chickens in Thailand.

Avibacterium paragallinarum causes infectious coryza in chickens, an acute respiratory disease that has worldwide economic significance. The objectives of this study were to determine the serovars, antimicrobial resistance, and pathogenicity of A. paragallinarum isolated from chickens in Thailand. Eighteen field isolates of A. paragallinarum were confirmed by PCR. When examined by serotyping in a hemagglutination inhibition test, 10 isolates were serovar A, five isolates were serovar B, and three isolates were serovar C. The susceptibility of the isolates to 16 antimicrobial agents was tested by a disk diffusion method. All isolates were susceptible to amoxicillin-clavulanic acid. There was a high level of resistance to lincomycin and erythromycin. All isolates were resistant to cloxacillin and neomycin. A study of bacterial entry into, and survival within, chicken macrophages showed variation between isolates but no clear connection to serovar. A virulence test was performed by challenging 4-wk-old layers via the nasal route with 400 dl of bacteria (10(8) colony-forming units/ml). Clinical signs were observed daily for 7 days, and the birds were subjected to a postmortem necropsy at 7 days postchallenge. All 18 field isolates caused the typical clinical signs of infectious coryza and could be re-isolated at 7 days after challenge. There was no significant difference in the clinical scores of the isolates except that two isolates (112179 and 102984, serovars A and B, respectively) gave a significantly higher score than did isolate CMU1009 (a serovar A isolate). No correlation between serovar and severity of clinical signs was found.