Problems can arise when vaccines and wild strains of a chicken herpesvirus recombine.

High‐frequency phase and antigenic variation of homologous lipoprotein haemagglutinins has been seen in both the major avian mycoplasma pathogens, Mycoplasma synoviae and Mycoplasma gallisepticum. The expression and, hence, antigenic variation of the pMGA gene family (encoding these lipoproteins in M. gallisepticum) is controlled by variation in the length of a trinucleotide repeat motif 5′ to the promoter of each gene. However, such a mechanism was not detected in preliminary observations on M. synoviae. Thus, the basis for control of variation in the vlhA gene family (which encodes the homologous haemagglutinin in M. synoviae) was investigated to enable comparison with its homologue in M. gallisepticum and with other lipoprotein gene families in mycoplasmas. The start point of transcription was identified 119 bp upstream of the initiation codon, but features associated with control of transcription in other mycoplasma lipoprotein genes were not seen. Comparison of three copies of vlhA revealed considerable sequence divergence at the 3′ end of the gene, but conservation of the 5′ end. Southern blot analysis of M. synoviae genomic DNA revealed that the promoter region and part of the conserved 5′ coding sequence occurred as a single copy, whereas the remainder of the coding sequence occurred as multiple copies. A 9.7 kb fragment of the genome was found to contain eight tandemly repeated regions partially homologous to vlhA, all lacking the putative promoter region and the single‐copy 5′ end of vlhA, but extending over one of four distinct overlapping regions of the 3′ coding sequence. Examination of sequential clones of M. synoviae established that unidirectional recombination occurs between the pseudogenes and the expressed vlhA, with duplication of pseudogene sequence and loss of the corresponding region previously seen in the expressed gene. Expression of the 5′ end of two variants of the vlhA gene showed that they differed in their reaction with monoclonal antibodies specific for this region. These data suggest that the control of vlhA antigenic variation in M. synoviae is achieved by multiple gene conversion events using a repertoire of coding sequences to generate a chimeric expressed gene, with the greatest potential for variation generated in the region encoding the haemagglutinin. Thus, completely distinct mechanisms have been adopted to control antigenic variation in homologous gene families.

Infectious laryngotracheitis (ILT) has been identified in most countries around the world and remains a threat to the intensive poultry industry. Outbreaks of mild to moderate forms of ILT are common in commercial layer flocks, while sporadic outbreaks of ILT in broiler flocks have also been recognized as an emerging problem in several countries. Examination of viral isolates using restriction fragment length polymorphism of polymerase chain reaction (PCR-RFLP) from individual ILTV genes has suggested that some of these outbreaks were caused by vaccine strains. In this study, PCR-RFLP of a number of ILTV genes/genomic regions including gE, gG, TK, ICP4, ICP18.5, and open reading frame (ORF) B-TK was used to examine a number of historical and contemporary Australian ILTV isolates and vaccine strains. PCR-RFLP of gE using restriction endonuclease EaeI failed to distinguish between any of the isolates including the vaccine strains. PCR-RFLP of gG, TK, and ORFB-TK using restriction endonucleases MspI and FokI, respectively, divided all the isolates into two groups. PCR-RFLP of ICP18.5 and ICP4 using restriction endonuclease HaeIII separated the isolates into three different groups with some field isolates only able to be distinguished from vaccine strains by PCR-RFLP of ICP18.5. A combination of groupings including gG, TK, ICP4, ICP18.5, and ORFB-TK PCR-RFLP classified the ILTV isolates under investigation into five different groups with most isolates distinguishable from vaccine strains. Results from this study reveal that to achieve reliable identification of strains of ILTV, the examination of multiple gene regions will be required, and that most of the recent ILT outbreaks in Australia are not being caused by vaccine strains.

ABSTRACT:Chlamydia infection is known to impact the health of koalas (Phascolarctos cinereus) in New South Wales (NSW) and Queensland, but the clinical significance of Chlamydia infections in Victorian koalas is not well described. We examined the prevalence of Chlamydia infection and assessed associated health parameters in two Victorian koala populations known to be Chlamydia positive. The same testing regimen was applied to a third Victorian population in which Chlamydia had not been detected. We examined 288 koalas and collected samples from the urogenital sinus and conjunctival sacs. Detection and differentiation of Chlamydia species utilized real-time PCR and high-resolution melting curve analysis. Chlamydia pecorum was detected in two populations (prevalences: 25% and 41%, respectively) but only from urogenital sinus swabs. Chlamydia was not detected in the third population. Chlamydia pneumoniae was not detected. Chlamydia pecorum infection was positively associated with wet bottom (indicating chronic urinary tract disease) in one Chlamydia-positive population and with abnormal urogenital ultrasound findings in the other Chlamydia-positive population. The prevalence of wet bottom was similar in all populations (including the Chlamydia-free population), suggesting there is another significant cause (or causes) of wet bottom in Victorian koalas. Ocular disease was not observed. This is the largest study of Chlamydia infection in Victorian koalas, and the results suggest the potential for epidemiologic differences related to Chlamydia infections between Victorian koalas and koalas in Queensland and NSW and also between geographically distinct Victorian populations. Further studies to investigate the genotypes of C. pecorum present in Victorian koalas and to identify additional causes of wet bottom in koalas are indicated.

Mycoplasma synoviae is an economically important pathogen of poultry worldwide, causing respiratory infection and synovitis in chickens and turkeys. Identification of M. synoviae isolates is of critical importance, particularly in countries in which poultry flocks are vaccinated with the live attenuated M. synoviae strain MS-H. Using oligonucleotide primers complementary to the single-copy conserved 59 end of the variable lipoprotein and haemagglutinin gene (vlhA), amplicons of~400 bp were generated from 35 different M. synoviae strains/isolates from chickens and subjected to mutation scanning analysis. Analysis of the amplicons by single-strand conformation polymorphism (SSCP) revealed 10 distinct profiles (A-J). Sequencing of the amplicons representing these profiles revealed that each profile related to a unique sequence, some differing from each other by only one base-pair substitution. Comparative high-resolution melting (HRM) curve analysis of the amplicons using SYTO 9 green fluorescent dye also displayed profiles which were concordant with the same 10 SSCP profiles (A-J) and their sequences. For both mutation detection methods, the Australian M. synoviae strains represented one of the A, B, C or D profiles, while the USA strains represented one of the E, F, G, H, I or J profiles. The results presented in this study show that the PCR-based SSCP or HRM curve analyses of vlhA provide high-resolution mutation detection tools for the detection and identification of M. synoviae strains. In particular, the HRM curve analysis is a rapid and effective technique which can be performed in a single test tube in less than 2 h.

Avian pathogenic Escherichia coli (APEC) cause widespread economic losses in poultry production and are potential zoonotic pathogens. Genome sequences of 95 APEC from commercial poultry operations in four Australian states that carried the class 1 integrase gene intI1, a proxy for multiple drug resistance (MDR), were characterized. Sequence types ST117 (22/95), ST350 (10/95), ST429 and ST57 (each 9/95), ST95 (8/95) and ST973 (7/95) dominated, while 24 STs were represented by one or two strains. FII and FIB repA genes were the predominant (each 93/95, 98 %) plasmid incompatibility groups identified, but those of B/O/K/Z (25/95, 26 %) and I1 (24/95, 25 %) were also identified frequently. Virulence-associated genes (VAGs) carried by ColV and ColBM virulence plasmids, including those encoding protectins [iss (91/95, 96 %), ompT (91/95, 96 %) and traT (90/95, 95 %)], iron-acquisition systems [sitA (88/95, 93 %), etsA (87/95, 92 %), iroN (84/95, 89 %) and iucD/iutA (84/95, 89 %)] and the putative avian haemolysin hylF (91/95, 96 %), featured prominently. Notably, mobile resistance genes conferring resistance to fluoroquinolones, colistin, extended-spectrum β-lactams and carbapenems were not detected in the genomes of these 95 APEC but carriage of the sulphonamide resistance gene, sul1 (59/95, 63 %), the trimethoprim resistance gene cassettes dfrA5 (48/95, 50 %) and dfrA1 (25/95, 27 %), the tetracycline resistance determinant tet(A) (51/95, 55 %) and the ampicillin resistance genes blaTEM-1A/B/C (48/95, 52 %) was common. IS26 (77/95, 81 %), an insertion element known to capture and mobilize a wide spectrum of antimicrobial resistance genes, was also frequently identified. These studies provide a baseline snapshot of drug-resistant APEC in Australia and their role in the carriage of ColV-like virulence plasmids.

These results indicate that PCR/HRM genotyping is a reliable diagnostic method for FAdV identification and is more rapid than virus neutralisation and direct sequence analysis. Furthermore, they suggest that IBH in Australian broiler flocks is a primary disease resulting from two alternative FAdV strains from different species.

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