C. J. Naylor

Exacerbation of Mycoplasma gallisepticum infection in turkeys by rhinotracheitis virus.

Groups of 1-day-old turkey poults from a parent flock free of antibodies to turkey rhinotracheitis virus (TRTV) and the pathogenic mycoplasmas, were infected by eyedrop with virulent TRTV, with Mycoplasma gallisepticum (Mg) or with both agents together. Dual infection resulted in increased morbidity compared with those groups given single infections. The presence of the Mg in the dual infection had no apparent effect on the pathogenesis of the virus, but the virus caused the Mycoplasma to be more invasive. Mg infection caused a transient depression in TRTV ELISA antibody titres at 29 days post-inoculation. At 14 days post-infection Mg haemagglutination inhibition (HI) and rapid serum agglutination (RSA) titres were higher (P <0.01) in the mixed infection group compared with those infected with Mg alone, but there was no significant difference between ELISA antibody titres of these two groups.

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.

Field avian metapneumovirus evolution avoiding vaccine induced immunity.

Live avian metapneumovirus (AMPV) vaccines have largely brought turkey rhinotracheitis (TRT) under control in Europe but unexplained outbreaks still occur. Italian AMPV longitudinal farm studies showed that subtype B AMPVs were frequently detected in turkeys some considerable period after subtype B vaccination. Sequencing showed these to be unrelated to the previously applied vaccine. Sequencing of the entire genome of a typical later isolate showed numerous SH and G protein gene differences when compared to both a 1987 Italian field isolate and the vaccine in common use. Experimental challenge of vaccinated birds with recent virus showed that protection was inferior to that seen after challenge with the earlier 1987 isolate. Field virus had changed in key antigenic regions allowing replication and leading to disease in well vaccinated birds.

Demonstration of a virulent subpopulation in a prototype live attenuated turkey rhinotracheitis vaccine

A prototype live attenuated turkey rhinotracheitis (TRT) vaccine, which was known to cause occasional disease in young poults following multiple back passage, was tested for the presence of a virulent subpopulation by a novel combined in vitro and in vivo screening technique. When vaccine was inoculated at high titres into chick embryo tracheal organ cultures, 17% of aliquots were found to cause ciliostasis, and when these aliquots, in turn, were inoculated into 1-day-old poults, approximately one-quarter caused clinical disease. Removal of the subpopulation by plaque purification led to viruses which had reduced tendency to revert to virulence but remained protective. The technique proved valuable in identifying virulent subpopulations in specific prototype TRT vaccines. The principle may have more general application.

Avian metapneumovirus (AMPV) attachment protein involvement in probable virus evolution concurrent with mass live vaccine introduction

Avian metapneumoviruses detected in Northern Italy between 1987 and 2007 were sequenced in their fusion (F) and attachment (G) genes together with the same genes from isolates collected throughout western European prior to 1994. Fusion protein genes sequences were highly conserved while G protein sequences showed much greater heterogeneity. Phylogenetic studies based on both genes clearly showed that later Italian viruses were significantly different to all earlier virus detections, including early detections from Italy. Furthermore a serine residue in the G proteins and lysine residue in the fusion protein were exclusive to Italian viruses, indicating that later viruses probably arose within the country and the notion that these later viruses evolved from earlier Italian progenitors cannot be discounted. Biocomputing analysis applied to F and G proteins of later Italian viruses predicted that only G contained altered T cell epitopes. It appears likely that Italian field viruses evolved in response to selection pressure from vaccine induced immunity.

Genetically diverse coronaviruses in wild bird populations of northern England.

Infectious bronchitis virus (IBV) causes a costly respiratory viral disease of chickens. The role of wild birds in the epidemiology of IBV is poorly understood. We detected diverse coronaviruses by PCR in wildfowl and wading birds in England. Sequence analysis showed some viruses to be related to IBV.

Microwave or autoclave treatments destroy the infectivity of infectious bronchitis virus and avian pneumovirus but allow detection by reverse transcriptase-polymerase chain reaction

A method is described for enabling safe transit of denatured virus samples for polymerase chain reaction (PCR) identification without the risk of unwanted viable viruses. Cotton swabs dipped in avian infectious bronchitis virus (IBV) or avian pneumovirus (APV) were allowed to dry. Newcastle disease virus and avian influenza viruses were used as controls. Autoclaving and microwave treatment for as little as 20 sec destroyed the infectivity of all four viruses. However, both IBV and APV could be detected by reverse transcriptase (RT)-PCR after autoclaving and as long as 5 min microwave treatment (Newcastle disease virus and avian influenza viruses were not tested). Double microwave treatment of IBV and APV with an interval of 2 to 7 days between was tested. After the second treatment, RT-PCR products were readily detected in all samples. Swabs from the tracheas and cloacas of chicks infected with IBV shown to contain infectious virus were microwaved. Swabs from both sources were positive by RT-PCR. Microwave treatment appears to be a satisfactory method of inactivating virus while preserving nucleic acid for PCR identification.

Failure of Maternal Antibodies to Protect Young Turkey Poults Against Challenge with Turkey Rhinotracheitis Virus

Groups of turkey poults with high levels of maternal antibodies (MA+) to turkey rhinotracheitis virus (TRTV) were challenged with virulent TRTV at 1, 5, and 10 days of age. A maternal antibody-free group (MA-) was also challenged at 1 day of age. Before each challenge, levels of maternal antibodies to TRTV were measured by enzyme-linked immunosorbent assay. Clinical signs were scored for each group. Unchallenged poults showed no signs. Respiratory signs in poults infected at 10 days of age resembled those seen in MA- birds infected at 1 day of age but both were more severe than in MA+ birds infected at 1 day of age, when the maternal antibodies were highest. However, overall, the presence of high levels of maternal antibodies did not prevent the development of clinical disease.

A turkey rhinotracheitis outbreak caused by the environmental spread of a vaccine-derived avian metapneumovirus

Avian metapneumovirus (aMPV) subtype A was isolated from 7-week-old turkeys showing respiratory disease typical of turkey rhinotracheitis. Comparison of the virus sequence with previously determined vaccine marker sequences showed that the virulent virus had originated from a licensed live subtype A aMPV vaccine. The vaccine had neither been in use on the farm within a period of at least 6 months nor had it been used on farms within a distance of approximately 5 km. Isolation of the virus and exposure to naive turkeys caused disease typical of a virulent aMPV field strain. The study shows that disease was caused by exposure to aMPV vaccine-derived virus that was present in the environment, and indicates that such virus is able to circulate for longer than was previously envisaged.

Duration of cross-protection between subtypes A and B avian pneumovirus in turkeys.

The degree and duration of clinical and virological cross-protection between avian pneumovirus subtypes A and B were examined in two-week-old pneumovirus antibody-free turkeys. The turkeys were inoculated with either a virulent subtype A (Belgian isolate AIT6/96), a virulent subtype B (Belgian isolate B/T9/96), an attenuated subtype A or an attenuated subtype B, and challenged homologously and heterologously with virulent avian pneumovirus two, five and 11 weeks after inoculation. Birds inoculated with virulent A or B virus showed typical respiratory signs from three to seven days after inoculation. After challenge, no clinical signs were observed in any of the groups, and no virus was isolated from the turkeys that had been initially inoculated with a virulent strain. Virulent virus was recovered from the birds that had been initially inoculated with attenuated subtypes and challenged five and/or 11 weeks later with a heterologous virulent strain. Birds challenged after five weeks showed a serological booster reaction only when they had been inoculated initially with a virulent or attenuated subtype B and challenged with subtype A. Seroconversion was observed in all the groups challenged after 11 weeks except when they had been inoculated initially with attenuated subtype B and challenged with subtype B.