Antimicrobial resistance (AMR): an important one health issue for layer and meat poultry industries worldwide.

Routine antibiotic administration has been used in intensive animal industries for a long time for health and production benefits. There is now a concerted effort to limit antibiotics administration to only treatment of clinically affected animals and to look for other alternative solutions combined with better husbandry practices for the benefits routine antibiotic administration seems to provide in intensive farming systems. In this paper it is argued that the benefits from routine antibiotics in chickens administration in lay are from suppression of the effects of mycoplasma infections. Mycoplasma freedom has been recommended but is not always practical. Vaccination of mycoplasma negative chickens with live mycoplasma vaccines is now being used (with biosecurity) to decrease antibiotic dependence in lay of poultry in many parts of the world.

Obg plays a significant role in temperature sensitivity of Mycoplasma synoviae live attenuated vaccine strain MS-H.

The MS-H vaccine strain (Vaxsafe MS®; Bioproperties Pty. Ltd., Australia) is a live attenuated temperature sensitive derivative of a virulent strain of M. synoviae, 86079/7NS, and is used to prevent diseases from M. synoviae challenges in poultry farms. The genome sequence of MS-H includes 32 single nucleotide polymorphisms (SNPs) compared to that of 86079/7NS. To investigate the nature of mutations responsible for temperature sensitivity, MS-H strain was subjected to thermal adaptation in vitro and in vivo. The only observed variation detected in the MS-H culture following sequential passages with incremental incubation temperature from 33 °C to 39.5 °C was an Ala210Val variation in Obg protein, associated with loss of temperature sensitivity phenotype. An identical variation was detected in the MS-H culture reisolated from one out of five bird 28 days after inoculation with MS-H. These findings suggest that M. synoviae is capable of thermoadaptive evolution and Obg plays a significant role in this trait.

Reversion of mutations in a live mycoplasma vaccine alters its metabolism.

The live attenuated temperature sensitive vaccine strain MS-H (Vaxsafe® MS, Bioproperties Pty. Ltd., Australia) is widely used to control disease associated with M. synoviae infection in commercial poultry. MS-H was derived from a field strain (86079/7NS) through N-methyl-N'-nitro-N-nitrosoguanidine (NTG)-induced mutagenesis. Whole genomic sequence analysis of the MS-H and comparison with that of the 86079/7NS have found that MS-H contains 32 single nucleotide polymorphisms (SNPs). Three of these SNPs, found in the obgE, oppF and gapdh genes, have been shown to be prone to reversion under field condition, albeit at a low frequency. Three MS-H reisolates containing the 86079/7NS genotype in obgE (AS2), obgE and oppF (AB1), and obgE, oppF and gapdh (TS4), appeared to be more immunogenic and transmissible compared to MS-H in chickens. To investigate the influence of these reversions in the in vitro fitness of M. synoviae, the growth kinetics and steady state metabolite profiles of the MS-H reisolates, AS2, AB1 and TS4, were compared to those of the vaccine strain. Steady state metabolite profiling of the reisolates showed that changes in ObgE did not significantly influence the metabolism, while changes in OppF was associated with significant alterations in uptake of peptides and/or amino acids into the M. synoviae cell. It was also found that GAPDH plays a role in metabolism of the glycerophospholipids as well as an arginine deiminase (ADI) pathway. This study underscores the role of ObgE, OppF and GAPDH in M. synoviae metabolism, and suggests that the impaired fitness arising from variations in ObgE, OppF and GAPDH contributes to attenuation of MS-H.

Characterization of atypical Mycoplasma anserisalpingitidis strains.

Mycoplasma anserisalpingitidis is a waterfowl colonizing mycoplasma, mainly found in geese. In this study, we compared the whole genomes of five atypical M. anserisalpingitidis strains originating from China, Vietnam and Hungary, with the rest of the collection. Common methods used in the description of species are genomic analyses like the analysis of 16 S - intergenic transcribed spacer (ITS) - 23 S rRNA, of housekeeping genes, of the average nucleotide identity (ANI) and average amino acid identity (AAI) and phenotypic analyses like testing the growth inhibition and the growth parameters of the strains. The atypical strains showed notable genomic differences in all of the genetic analyses: on average ANI and AAI 95% (M. anserisalpingitidis ANI Minimum: 92.45, Maximum: 95.10; AAI Minimum: 93.34, Maximum: 96.37). The atypical strains formed a separate branch among the M. anserisalpingitidis strains in all phylogenetic studies. The small genome size and possibly higher mutation rate of the M. anserisalpingitidis species likely contributed to the observed genetic difference. Based on genetic analyses, the studied strains clearly represent a new genotype of M. anserisalpingitidis. The atypical strains showed slower growth in the medium containing fructose and three of the atypical strains showed diminished growth in the inhibition test. However, no definitive geno-phenotype associations were found regarding the fructose metabolism pathway in the atypical strains. The atypical strains are potentially at an early stage of speciation.

Effects of administration of tylosin on the duration of protective immunity induced by the novel Mycoplasma gallisepticum vaccine strain, Vaxsafe MG ts-304, in chickens.

Prophylactic use of antimicrobials after administration of live vaccines is a common practice in the poultry industry, but the impact of this on the efficacy and duration of protection induced by the vaccines is unknown. The effect of treatment with tylosin on the efficacy of vaccination with the live attenuated M. gallisepticum strain, Vaxsafe MG ts-304, was examined. This vaccine has previously been shown to provide protection for at least 57 weeks. Ten-week-old specific-pathogen-free chickens were vaccinated with Vaxsafe MG ts-304 and then treated with tylosin at a therapeutic dose in drinking water from 6 weeks after vaccination. Tylosin was withdrawn 5 days before challenge with M. gallisepticum strain Ap3AS at 6, 10, 14, 18 or 22 weeks after vaccination. Air sac lesions, tracheal mucosal thickening and the concentrations of serum antibodies against M. gallisepticum were assessed at 2 weeks after challenge. The protection induced by the vaccine in the 6 weeks before initiation of tylosin treatment persisted for 18 weeks after vaccination, with lesions only observed in the air sacs of vaccinated birds that had been treated with tylosin after challenge at 22 weeks after vaccination. Concentrations of serum antibodies against M. gallisepticum began to decrease in vaccinated birds that had been treated with tylosin from 16 weeks after vaccination. This study has suggested that treatment of chickens with tylosin after vaccination with a live attenuated mycoplasma vaccine reduces the duration of protective immunity afforded by the vaccine.

Virulence factors of Mycoplasma synoviae: Three genes influencing colonization, immunogenicity, and transmissibility.

Infections caused by Mycoplasma synoviae are major welfare and economic concerns in poultry industries worldwide. These infections cause chronic respiratory disease and/or synovitis in chickens and turkeys leading to reduced production and increased mortality rates. The live attenuated vaccine strain MS-H (Vaxsafe® MS), commonly used for protection against M. synoviae infection in many countries, contains 32 single nucleotide variations compared to its wildtype parent strain, 86079/7NS. Genomic analysis of vaccine strains reisolated from flocks following the administration of MS-H has identified reversions to the original 86079/7NS sequence in the obgE, oppF and gapdh genes. Here, three MS-H field reisolates containing the 86079/7NS genotype in obgE (AS2), obgE and oppF (AB1), and obgE, oppF and gapdh (TS4), as well as the vaccine MS-H and the parental strain 86079/7NS were experimentally inoculated to chickens. The strains were assessed for their ability to infect and elicit immune responses in the recipient chickens, as well as in naïve in-contact chickens. Despite the loss of temperature sensitivity phenotype and colonization of the reisolates in the lower respiratory tract, there was no significant differences detected in the microscopic mucosal thickness of the middle or lower trachea of the inoculated chickens. Concurrent reversions in ObgE, OppF and GAPDH proteins were associated with higher gross air sac lesion scores and increased microscopic upper-tracheal mucosal thickness in chickens directly inoculated with the reisolates following intratracheal administration of a virulent strain of infectious bronchitis virus. The gross air sac lesions of the chickens in-contact with those inoculated with reisolates were not significantly different to those of chickens in-contact with MS-H inoculated chickens, suggesting that horizontal transmission of the reisolates in the poultry flock will not lead to higher pathogenicity or clinical signs. These results suggest a significant role of GAPDH and/or cumulative effect of ObgE, OppF and GAPDH on M. synoviae pathogenicity. Future experiments will be required to investigate the effect of single mutations in gapdh or oppF gene on pathogenicity of M. synoviae.

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.

Antimicrobial susceptibility of pathogenic mycoplasmas in chickens in Asia.

Mycoplasma synoviae (n = 26) and M. gallisepticum (n = 11) isolates were gained from 164 clinical samples collected from China, India, Indonesia, Malaysia, Philippines, Republic of Korea and Thailand. Most isolates were from commercial chicken production systems. A method of filtering (0.45 µm) samples immediately after collection was convenient allowing over a week for transit to the laboratory. Minimum inhibitory concentrations (MICs) were characterized by a broth microdilution method to enrofloxacin, difloxacin, oxytetracycline, chlortetracycline, doxycycline, tylosin, tilmicosin, tylvalosin, tiamulin, florfenicol, lincomycin, spectinomycin and lincomycin and spectinomycin combination (1:2). Increased MICs to various antimicrobials were seen in different isolates but appeared largely unrelated to the antimicrobial treatment histories. Overall, the results were similar to other MIC surveys around the world. Generally, low MICs to tetracyclines, tiamulin and tylvalosin were observed. Increased tilmicosin MICs were observed in both M. synoviae and M. gallisepticum isolates (≥64 µg/ml MIC90 values) and this was seen in all isolates with high tylosin MICs. Increases in lincomycin MICs were mostly associated with increases in tilmicosin MICs. The results also suggested that antimicrobial use after mycoplasma vaccination may interfere with vaccine strain persistence and efficacy (field strains were more commonly observed in flocks that had treatments after vaccination) and this area warrants more investigation. The study shows that isolation and MIC determination can be done from remote locations and suggests that this may provide information that will allow more effective use of antimicrobials or other methods of control of avian mycoplasma in chickens (e.g. live vaccines) and therefore more responsible use of antimicrobials from a one health perspective.

Fatal skull trauma in caged layer chickens associated with a moving feed hopper: diagnosis based on autopsy examination, forensic computed tomography and farm visit.

Investigation of unexpected mortality in caged layer chickens led to the discovery of a consistent traumatic injury to the heads of affected hens. Initial post-mortem examination found linear skin lacerations and associated fractures in the dorsal cranium of all birds examined, and 5 to 10 mm deep trauma in the underlying brain tissue. Post-mortem multidetector computed tomography (CT) scanning of two affected birds demonstrated similar obliquely orientated, linear, depressed fractures of the skulls consistent with a single, severe impact force to the head. Both skull fractures had a pattern of rounded, rostral expansion measuring approximately 3 mm in width. On inspection of the cages during a farm visit, this CT pattern corresponded with the size and shape of sheet metal lugs holding feed troughs onto the cages (on which blood stains were subsequently observed). Based on this analysis and hypothesizing that hunger was a triggering factor, a recommendation was made to reverse the shed "lights on" and feed hopper operation times with instant reduction in mortality. This case highlights the value of post-mortem CT imaging in bird death investigation where trauma is a postulated cause.

Genotyping of Japanese field isolates of Mycoplasma synoviae and rapid molecular differentiation from the MS-H vaccine strain.

Mycoplasma synoviae is an important causative agent of avian mycoplasmosis. In the present study the conserved domain of the variable lipoprotein and hemagglutinin (vlhA) gene of M. synoviae was sequenced and analyzed for 19 field strains of M. synoviae isolated from chickens across Japan. This analysis revealed that there were at least nine genotypes of M. synoviae present in Japan. Furthermore, we found a single nucleotide polymorphism (SNP) within this region in all the Japanese isolates, and based on this finding, we established a PCR method with cycling probe technology to differentiate between these field isolates and the live M. synoviae vaccine strain Mycoplasma synoviae-H (MS-H).

Sequence analysis and comparison of avian hepatitis E viruses from Australia and Europe indicate the existence of different genotypes.

Avian hepevirus infections were detected in chickens suffering from big liver and spleen disease or hepatitis-splenomegaly syndrome in Australia, the USA and Europe. Available data indicate their genetic relationship to mammalian hepatitis E virus (HEV). In the present study, the near-complete genomic sequences of an Australian and a European isolate of avian hepatitis E virus (avian HEV) are reported for the first time. Furthermore, the phylogenetic relationship to other avian HEVs is determined. Sequence analyses of these isolates identified major genetic differences among avian HEVs. Most of them are located within the open reading frame (ORF)1 region, although only a few lie within conserved motifs of predicted domains. Non-silent mutations in the ORF2 region suggest the presence of potentially different epitopes among avian HEV isolates. Finally, phylogenetic analysis confirmed the distant relationship to mammalian HEV and additionally suggested that the avian HEVs can be separated into three different genotypes: 1 (Australia), 2 (USA) and 3 (Europe), indicating a geographical distribution pattern.

Avian hepatitis E virus infection and possible associated clinical disease in broiler breeder flocks in Hungary.

In broiler breeder flocks in one broiler integration in Hungary, a new syndrome appeared in January 2005 with initially four successive post-peak flocks experiencing significant decreases in egg production. Clinically birds became depressed and there was a small increase in the mortality rate. Postmortem examinations revealed enlarged livers in up to 19% of birds dying, and enlarged spleens in some. Also observed were birds with either clotted blood or serosanguineous fluid in the abdomen and subcapsular haemorrhages of the liver. Histopathology and polymerase chain reaction excluded tumours and the presence of common tumour-associated viruses. Chronic bacterial infections (especially causing hepatitis, peritonitis and airsacculitis) were common but many enlarged livers had no obvious bacterial involvement. After a 9-month period during which a majority of flocks became affected, no newly affected flocks occurred. Investigations showed that all tested affected flocks were seropositive in the big liver and spleen (BLS) Agar Gel Immunodiffusion test. Subsequent flocks without post-peak egg-production drops were shown to be seronegative in the BLS AGID test, as were all the parent flocks contributing to the affected flocks. Liver samples and cloacal swabs were positive by polymerase chain reaction (aHEV helicase target), and calicivirus-like particles were demonstrated in bile samples from affected birds. These observations are similar to hepatitis-splenomegaly syndrome as described in North America and BLS syndrome as described in Australia. Histopathological features were a non-specific chronic hepatitis similar to those described in BLS and hepatitis-splenomegaly syndrome. Immunohistochemistry using a BLS-specific monoclonal antibody confirmed the presence of avian hepatitis E virus antigen in livers and spleen.

Identification and sequencing of the groE operon and flanking genes of Lawsonia intracellularis: use in phylogeny.

Proliferative enteropathy (PE) is a complex of diseases of commercial importance to the pig industry. The obligate intracellular bacterium Lawsonia intracellularis is consistently associated with PE and pure cultures of this bacterium have been used to reproduce PE in pigs. In this study L. intracellularis bacteria were purified directly from PE-affected tissue. DNA extracted from purified bacteria was used to construct a partial genomic library which was screened using sera from L. intracellularis-immunized rabbits. Two seroreactive recombinant clones were identified, one of which expressed proteins of 10 and 60 kDa. The sequence of the insert from this clone, pISI-2, revealed ORFs with sequence similarity to the groES/EL operon of Escherichia coli, the 505 ribosomal proteins L21 and L27 of E. coli, a GTP-binding protein of Bacillus subtilis and a possible protoporphyrinogen oxidase, HemK, of E. coli. Primers designed from unique sequences from the pISI-2 insert amplified DNA from infected, but not non-infected, porcine ilea; the amplicon sequence obtained from tissue-cultured L. intracellularis was identical to the corresponding sequence in pISI-2, confirming the origin of the clone. The sequence of L. intracellularis GroEL and other GroEL sequences in the databases were used to construct a partial phylogenetic tree. Analysis of the GroEL sequence relationship suggested that L. intracellularis is not significantly related to other organisms whose GroEL sequences are held in the databases and supports previous data from 16S sequence analyses suggesting that L. intracellularis is a member of a novel group of enteric pathogens.