Karen J. Worthington

A reverse transcriptase-polymerase chain reaction survey of infectious bronchitis virus genotypes in Western Europe from 2002 to 2006

A survey of infectious bronchitis virus (IBV) genotypes in poultry flocks in selected countries in Western Europe was carried out between March 2002 and December 2006. Identification of IBV was by reverse transcriptase-polymerase chain reaction of RNA extracted from oropharyngeal swabs taken from poultry flocks exhibiting signs of clinical disease thought to be attributable to IBV. Part of the hypervariable S1 gene of IBV was sequenced to differentiate between the various genotypes. During the survey, 4103 samples were processed, of which 2419 (59%) were positive for IBV. The predominant IBV genotypes detected were 793B and Massachusetts. The third and fourth most common genotypes were two new economically important field types: Italy02, and a virus similar to genotypes originally detected in China called QX. Analysis of the partial S1 sequences of the genotypes detected suggested that approximately 50% of all 793B, Massachusetts types and D274 IBVs were identical to the homologous commercially available live vaccines. Since 2004 the prevalence of Italy02 (present in all countries from which samples were received) has been declining in all countries except Spain, where it appeared to be the predominant genotype. Since 2004 an IBV genotype has been detected in Holland, Germany, Belgium and France similar to QX and the incidence has increased. QX was not detected in the United Kingdom or Spain. When detections thought to be attributable to vaccines were removed, the dominant genotype in France and Europe overall was 793B; in Germany, Holland and Belgium, it was QX-like IBV; and in the United Kingdom and Spain the dominant genotype was Italy02. The present study is the first to identify the prevalence of both Italy02 and QX field-type variants in poultry flocks in Western Europe. Several novel genotypes have also been detected.

Chinese QX strain of infectious bronchitis virus isolated in the UK

SIR, — We wish to report the isolation and identification of a strain of infectious bronchitis virus (ibv) which shows a close genetic relationship with the so-called Chinese qx strain of ibv. As far as we are aware, this is the first report of the presence of this virus in the uk. A six- to

Effect of cyclophosphamide immunosuppression on the immunity of turkeys to viral rhinotracheitis

Turkey poults, free of antibodies to turkey rhinotracheitis (TRT) virus were treated with cyclophosphamide on days 1, 2 and 3 after hatching and vaccinated by eyedrop when 10 days old with a Vero cell-attenuated preparation of TRT virus. No ELISA antibodies to TRT virus developed in the sera of these poults but they were as resistant to virulent virus challenge 21 days later as vaccinated groups which were not cyclophosphamide-treated but produced humoral antibodies. Following challenge with virulent virus at 31 days old cyclophosphamide-treated unvaccinated poults developed a more severe clinical response than untreated birds and had higher virus titres in tracheal swabs. The findings show that the respiratory tract of turkeys may be resistant to TRT despite the absence of ELISA antibodies in the serum.

Further studies on the development of a live attenuated vaccine against turkey rhinotracheitis.

Turkey rhinotracheitis (TRT) virus attenuated by passaging in Vero cells was tested at two different passage levels (15 or 25 passages) and two dose levels [10(3) or 10(4) TCID50 (50% tissue culture infectious doses) per bird] to determine the efficacy in protecting turkey poults against experimental challenge with virulent TRT virus. Following administration by the eyedrop route at 10 days of age, all four preparations proved successful in providing protection against clinical disease and establishment of challenge virus in the trachea when challenged with virulent virus 3 weeks later. Twelve-day-old poults given the 25th Vero passage TRT virus at a dose of 10(3.5) TCID50 per bird were protected against experimental challenge with virulent virus for at least 22 weeks post-primary inoculation. The 25th passage virus was tested for safety by administering ten times the dose (10(4.5) TCID50 per bird) used in the previous trial to a group of 10-day-old turkey poults. None of the birds showed any clinical signs during 21 days post-inoculation. Attempts to back-passage the virus from bird to bird were unsuccessful.

Avian pneumovirus infection in broiler chicks inoculated with Escherichia coli at different time intervals

The effects of dual infection of 1-day-old broiler chicks with a chicken isolate of avian pneumovirus (APV) and a pool of pathogenic Escherichia coli strains were studied by supraconjunctival application of the bacteria simultaneously with the virus, or at 4, 7 or 11 days afterwards. When the agents were given together, the clinical disease was significantly more severe than that caused by the virus alone, but when the bacterium was given later the signs were less severe. None of the infections resulted in swollen head syndrome by 32 days. All mixed infections caused moderate to severe congestion in the turbinates, when birds were examined at 32 days of age, at which time no such lesions were present in birds having been infected with APV alone. E. coli was isolated from almost 100% of birds with mixed infections, while rates of those given only E. coli isolation varied between 56 and 67%. Furthermore, E. coli colony counts were consistently higher from mixed infection groups. Virus persistence in the choanal cleft was slightly prolonged in birds with the simultaneous mixed infection. Although the pool of E. coli included O2, O78 and O18 serotypes, only those of the O2 serotype and a small number of untypable strains were re-isolated from selected mixed and single E. coli-infected groups. Mixed APV and E. coli infection did not affect APV enzyme-linked immunosorbent assay antibody titres at 21 or 32 days. Thus, experimental infection of broiler chicks with APV and E. coli, simultaneously or at intervals afterwards, demonstrated a synergistic effect between the two agents, but none of the infection protocols caused swollen head syndrome.

A study on the kinetics of pancreatic kallikrein with the substrate benzoyl arginine ethyl ester using a spectrophotometric assay

Abstract A commercial preparation of kallikrein from pig pancreas was used to determine the optimum conditions for kallikrein estimation by measuring its esterolytic activity with the substrate benzoyl arginine ethyl ester using a spectrophotometric assay. Cysteine (1 mM) and EDTA (0.1 mM and 0.8 mM) significantly increased activity, thioglycollate (1 mM) had no effect, sodium chloride (100 mM) and triethanolamine (100 mM) both significantly decreased activity when added to 67 mM triethanolamine buffer, pH 8.0. The optimal triethanolamine concentration was 10 to 50 mM. The optimal pH was 8.5 to 9.0 and below pH 5.5 acid denaturation of the enzyme started to occur. The Michaelis constant K m was calculated to be 0.101 mM at pH 8.5. There appeared to be two basic groups in the enzyme and the enzyme substrate complex with pKs of 7.9 and 7.07 at 25°, respectively, which were involved in the reaction. The quantitative estimation of kallikrein using the alcohol dehydrogenase linked method was examined and modifications made to reduce the errors involved, enabling the spontaneous breakdown of substrate and the enzymatic breakdown to be measured simultaneously. This then provided a reproducible method for the estimation of impure solutions of kallikrein.