Development of a duplex qPCR for the differentiation of a live attenuated Escherichia coli aroA mutant vaccine strain from field isolates in chickens.

Avian pathogenic Escherichia coli (APEC) can cause colibacillosis in poultry, characterised by localised or systemic infections. Colibacillosis is considered one of the leading causes of economic losses in the poultry industry due to reduced performance, increased mortality, treatment costs and carcass condemnations. A live attenuated Escherichia coli O78 aroA gene mutant is widely used to prevent disease. However, no effective strategies to differentiate the vaccine strain from field strains are available, hampering follow-up of vaccination campaigns. In the current study, we report a PCR-based method to simultaneously detect the vaccine strain by targeting the vaccine-specific mutation in the aroA gene, as well as the wild type E. coli strains by targeting the xanQ gene. The specificity of this PCR was evaluated using 123 E. coli isolates, form which 5 WT aroA auxotrophic strains (WT strains with a natural aroA deficiency), as well as 7 non-Escherichia isolates. The PCR showed 100% sensitivity of the xanQ primers for E. coli detection and 100% sensitivity of the ΔaroA primers for the vaccine strain. In order to allow quantification of the vaccine strain in complex samples containing many different E. coli strains and other related organisms, such as chicken faeces, a probe-based duplex qPCR was developed. The limit of detection (LOD) of this duplex qPCR method was 8.4*103 copies/g faeces. The specificity of the duplex qPCR was confirmed by determining both the vaccine strain levels, and the total E. coli load in intestinal digesta from both vaccinated and non-vaccinated birds. E. coli could be detected in both vaccinated and non-vaccinated birds. The duplex qPCR was specific for the vaccine strain as this strain was detected in all vaccinated birds, whereas no signal was detected in non-vaccinated birds. The duplex qPCR is helpful in monitoring colonization and shedding of the vaccine strain.

Efficacy, Safety, and Interactions of a Live Infectious Bursal Disease Virus Vaccine for Chickens Based on Strain IBD V877.

Infectious bursal disease (IBD) is a highly contagious disease in young chickens which can result in high morbidity and mortality and also in great economic losses. The main target for the virus is the lymphoid tissue with a special predilection for the bursa of Fabricius. Several vaccines are available to control the disease. Intermediate plus vaccines are used in chickens with high maternal antibody titers which face high infection pressure. An example of an intermediate plus vaccine is a live vaccine based on IBD strain V877. The results of an efficacy study in commercial broilers with different levels of maternally derived antibodies (MDA) showed that the V877-based IBD vaccine can break through maternal antibody titers of higher than 1100 as determined by an IBD ELISA. The safety of the vaccine was demonstrated in a study in which specific-pathogen-free (SPF) chickens were vaccinated with a tenfold dose of the vaccine strain and a tenfold dose of the vaccine strain after five back passages in SPF chickens. The vaccine virus caused lesions, as could be expected for an intermediate plus vaccine, but the scores were not much higher than the maximal scores allowed for mild IBD vaccines in the European Pharmacopoeia, and reversion to virulence was absent. In studies in SPF chickens, there were no negative impacts by the IBD V877 vaccine on the efficacy of a live QX-like IB vaccine and a live Newcastle disease La Sota vaccine in vaccination challenge studies, although the IBD vaccine had a negative effect on the antibody response generated by the QX-like IB vaccine. It is concluded that the IBD V877 vaccine has the capacity to break through high levels of MDA, has a satisfactory safety profile, and interactions with other live vaccines are limited. In order to limit bursal lesions after vaccination it is recommended to confirm the presence of MDA before vaccinating with the V877 vaccine.

Multiplex PCR assay for the detection of high virulence rabbit Staphylococcus aureus strains.

High virulence rabbit Staphylococcus aureus strains, which are clonal in origin, are responsible for the spread of chronic staphylococcosis at the rabbit flock level. The aim of the present study was to develop a multiplex PCR assay that can be used for the identification of these high virulence strains. Two targets of the assay were the bbp and the selm genes, which have recently been shown to occur specifically in high virulence isolates. A third target was a sequence designated "flank", which was derived from a previously generated high virulence specific RAPD pattern. Furthermore, the femA gene, which is specific for S. aureus, was incorporated in order to avoid false negative results due to insufficient DNA preparation. The multiplex PCR was successful at differentiating the 26 typical high virulence and 50 low virulence rabbit S. aureus strains incorporated in the present study. Therefore it is useful for the initial screening of newly acquired breeding stock, in order to prevent the intake of high virulence strains in rabbitries.

Prevalence of genes encoding exfoliative toxins, leucotoxins and superantigens among high and low virulence rabbit Staphylococcus aureus strains.

Staphylococcus aureus is an important cause of pododermatitis, subcutaneous abscesses and mastitis in rabbits. On rabbitry level, two types of S. aureus infections can be distinguished. In the first type, caused by low virulence strains, the infection affects only a small number of animals. The second type of infection is caused by high virulence strains and spreads throughout the rabbit flock. The pathogenic capacity of a particular S. aureus strain is attributed to a combination of invasive properties and extracellular factors such as toxin production. Therefore, 22 high virulence and 37 low virulence S. aureus isolates were compared regarding the prevalence of genes encoding exfoliative toxins, leucotoxins and superantigens. This study revealed a clearly significant difference between HV and LV rabbit S. aureus strains. All typical HV isolates were positive for the egc cluster, containing the enterotoxin(like) genes seg, sei, selm, seln, selo and selu, whereas these genes were not detected in any of the LV isolates. Further research is necessary to clarify the importance of the egc cluster in the pathogenesis of infections with high virulence S. aureus strains in rabbits.

Genotypic and phenotypic screening of high and low virulence Staphylococcus aureus isolates from rabbits for biofilm formation and MSCRAMMs.

At rabbit flock level, two types of Staphylococcus aureus infections can be distinguished. In the first type, caused by low virulence strains, the infection remains limited to a small number of animals. The second type of infection is caused by the high virulence strains, which spread throughout the rabbitry. The pathogenetic capacity of a particular S. aureus strain is attributed to a combination of extracellular factors and invasive properties such as adherence and biofilm formation. Twenty eight high virulence and 34 low virulence S. aureus isolates recovered from rabbits between 1998 and 2003 were used to study slime production on Congo red Agar (CRA) and prevalence of bap, icaA and icaD associated with biofilm formation. Furthermore these strains were screened for the presence of bbp, clfA, clfB, cna, ebpS, eno, fnbA, fnbB and fib encoding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). The presence of icaA and icaD was not correlated with slime production on CRA. Bap was absent in all strains. All rabbit S. aureus strains harboured clfA and clfB. The prevalences of ebpS, eno, fnbA and fib did not reveal striking differences between high and low virulence strains. FnbB prevalence in high virulence isolates was lower than in low virulence isolates and cna was absent in high virulence strains. It was remarkable that only high virulence strains were positive for bbp. Further research is necessary to elucidate the significance of bbp in the pathogenesis of high virulence strains.

Antimicrobial resistance and resistance genes in Staphylococcus aureus strains from rabbits.

Fifty-six Staphylococcus aureus isolates recovered between 1998 and 2003 from 31 rabbit farms with and without problems of chronic staphylococcosis, were screened for resistance to enrofloxacin, erythromycin, gentamicin, lincomycin, neomycin, penicillin and tetracyclines using the agar dilution test. For penicillin, a disk diffusion test was also performed. The detection of tetP(B), tet(K), tet(L), tet(M), tet(O), tet(T), tet(W), erm(A), erm(B), erm(C) and mec(A) genes was done via a PCR assay. Four isolates showed resistance to erythromycin and lincomycin. These isolates were positive for the erm(C) gene in the PCR. Eleven strains were resistant to tetracyclines and all harboured the tet(K) gene. In the agar dilution test, five isolates showed resistance to penicillin, whereas in the disk diffusion test 12 isolates showed resistance. None of these 12 resistant isolates carried the mec(A) gene. Only one strain showed resistance to gentamicin, and all strains were susceptible to enrofloxacin and neomycin. This study demonstrates that resistance to antimicrobial agents in S. aureus isolates originating from rabbits is relatively rare compared to resistance in S. aureus isolates originating from other animals and humans.