E. Catelli

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.

The use of virus isolation, histopathology and immunoperoxidase techniques to study the dissemination of a chicken isolate of avian pneumovirus in chickens

A subgroup B isolate of turkey rhinotracheitis virus (TRTV) or avian pneumovirus (APV), obtained from a flock of commercial breeding chickens experiencing poor egg production, mortality and swollen head syndrome, was shown to cause substantial respiratory signs in both young SPF chickens and chicks with high levels of maternally derived TRT antibodies. This isolate replicated to high titre in the respiratory tract of experimentally inoculated SPF chickens for approximately 5 days after inoculation, but was recovered only occasionally after that time. It was never recovered from non-respiratory tract tissues. A detailed, sequential histological and immunoperoxidase study was performed. This revealed that, whilst TRT virus could be demonstrated consistently in the epithelium of upper respiratory tract tissue, although only for a short time after inoculation, the damage which it caused was minimal and recovery was rapid. This study, using a pathogenic TRT isolate obtained from diseased chickens, provides clear evidence that TRT virus can cause damage to the respiratory tract of chickens and that this damage is both localized and short lived.

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.

Ecological Aspects of Influenza A Virus Circulation in Wild Birds of the Western Palearctic

M. Delogu1*, M.A. De Marco2, I. Donatelli3, L. Campitelli3 and E. Catelli1 1Dipartimento di Sanita Pubblica Veterinaria e Patologia Animale, Facolta di Medicina Veterinaria, Universita degli Studi di Bologna, Ozzano Emilia (BO); 2Istituto Nazionale per la Fauna Selvatica, Ozzano Emilia (BO); 3L aboratory of V irology, Istituto Superiore di Sanita, Rome, Italy. *Correspondence: Dipartimento di Sanita Pubblica Veterinaria e Patologia Animale, Facolta di Medicina Veterinaria, Universita degli Studi di Bologna, 50 via T olara di Sopra, 40064 Ozzano Emilia (BO), Italy E-mail: delogu@vet.unibo.it

In vitro antiviral activity of chestnut and quebracho woods extracts against avian reovirus and metapneumovirus.

Field evidences have suggested that a natural extract, containing tannins, could be effective against poultry enteric viral infections. Moreover previous studies have shown that vegetable tannins can have antiviral activity against human viruses. Based on this knowledge three different Chestnut (Castanea spp.) wood extracts and one Quebracho (Schinopsis spp.) wood extract, all containing tannins and currently used in the animal feed industry, were tested for in vitro antiviral activity against avian reovirus (ARV) and avian metapneumovirus (AMPV). The MTT assay was used to evaluate the 50% cytotoxic compounds concentration (CC(50)) on Vero cells. The antiviral properties were tested before and after the adsorption of the viruses to Vero cells. Antiviral activities were expressed as IC(50) (concentration required to inhibit 50% of viral cytopathic effect). CC(50)s of tested compounds were > 200 microg/ml. All compounds had an extracellular antiviral effect against both ARV and AMPV with IC(50) values ranging from 25 to 66 microg/ml. Quebracho extract had also evident intracellular anti-ARV activity (IC(50) 24 microg/ml). These preliminary results suggest that the examined vegetable extracts might be good candidates in the control of some avian virus infections. Nevertheless further in vivo experiments are required to confirm these findings.

Pediocin A improves growth performance of broilers challenged with Clostridium perfringens

The aim of this study was to investigate the efficacy of the anticlostridial pediocin A from Pediococcus pentosaceus FBB61 to contain negative effects associated to Clostridium proliferation in broilers, through 2 subsequent investigations. In the first study, 36 Ross 508 broilers were divided into 3 groups and fed for 21 d as follows: the control diet (CTR), the control diet supplemented with supernatant filtrate of a culture of P. pentosaceus FBB61-2 (Bac-, isogenic mutant nonproducing pediocin A), and the control diet supplemented with supernatant filtrate of a culture of P. pentosaceus FBB61 (Bac+) containing pediocin A. Birds were challenged with 10(6) cells of Clostridium perfringens. In the second study, 216 Ross 508 broilers were allocated in 18 pens and divided into 3 groups fed the same diet for 42 d: a control group (CTR), a group challenged with 10(8) cells of C. perfringens (CP), and a group challenged with 10(8) cells of C. perfringens and receiving the control diet supplemented with P. pentosaceus FBB61 and pediocin A (PA). Broiler BW, ADG, ADFI, and feed conversion rate were measured throughout the studies. At the end of both experiments, an appropriate number of birds was killed and analyzed for necrotic enteritis lesions and microbiological examinations. In the first study, on d 9, ADG and BW were 20% higher for Bac+ compared with CTR and Bac-; on d 14, ADG was higher for Bac+ (+23%, P<0.05), whereas BW was higher for Bac+ and Bac- compared with CTR (+23 and +14%, respectively; P<0.05). In the second study, on d 14, ADG and BW were higher for PA compared with CTR and CP (+15% on average; P<0.05), whereas between 15 and 42 d, there was only a tendency toward a higher ADG for PA when compared with the CP group (+4%, P=0.08). Diet supplementation with pediocin A improved broiler growth performance during the challenge with C. perfringens and tended to restore the ADG depletion during the 42-d period.

Serological evidence of avian pneumovirus infection in reared and free-living pheasants

The study was funded by the French Ministry of Agriculture and Fisheries, and followed by a scientific steering committee (Marc Girard, CERVI, Lyon, France, Jeanne Brugere-Picoux, ENVA, Maisons-Alfort, France, Dominique Costagliola, INSERM SC4, Paris, France, Jean-Claude Desenclos, INVS, SaintMaurice, France, Marc Eloit, ENVA, Maisons-Alfort, France, Dagmar Heim, Office veterinaire federal, Liebefeld, Switzerland, Francois Moutou, AFSSA, Maisons-Alfort, France, Annick Tibi, Pharmacie centrale des h6pitaux, Paris, France). The authors would especially like to thank J-N. Arsac, N. Besson, E. Charmette, S. Dechavanne and L. Maitrias (AFSSA Lyon) for their excellent technical assistance, and F. Gauchard and S. La Vieille (AFSSA Derns) for their efficient and valuable assistance in scientific coordination. The study was made feasible by the involvement of the veterinary practitioners, the CVLS, the county veterinary services and the Direction generale de lalimentation of the MAF in charge of the overall logistics of the study.

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.

Development of a real-time RT-PCR assay for the simultaneous identification, quantitation and differentiation of avian metapneumovirus subtypes A and B

In recent years, special attention has been paid to real-time polymerase chain reaction (PCR) for avian metapneumovirus (AMPV) diagnosis, due to its numerous advantages over classical PCR. A new multiplex quantitative real-time reverse transcription-PCR (qRT-PCR) with molecular beacon probe assay, designed to target the SH gene, was developed. The test was evaluated in terms of specificity, sensitivity and repeatability, and compared with conventional RT nested-PCR based on the G gene. All of the AMPV subtype A and B strains tested were amplified and specifically detected while no amplification occurred with other non-target bird respiratory pathogens. The detection limit of the assay was 10−0.41 median infectious dose/ml and 101.15 median infectious dose/ml when the AMPV-B strain IT/Ty/B/Vr240/87 and the AMPV-A strain IT/Ty/A/259-01/03 were used, respectively, as templates. In all cases, the amplification efficiency was approximately 2 and the error values were <0.2. Standard curves, generated either using the serial dilution of an RNA suspension or RNA extracted from the serial dilution of titrated viral suspensions as templates, exhibited good linearity (R 2>0.9375) between crossing point values and virus quantities, making the assay herein designed reliable for quantification. When the newly developed qRT-PCR was compared with a conventional RT nested-PCR, it showed greater sensitivity with RNA extracted from both positive controls and from experimentally infected birds. This assay can be effectively used for the detection, identification, differentiation and quantitation of AMPV subtype A or subtype B to assist in disease diagnosis and to carry out rapid surveillance with high levels of sensitivity and specificity.

A single polymerase (L) mutation in avian metapneumovirus increased virulence and partially maintained virus viability at an elevated temperature

Previously, a virulent avian metapneumovirus, farm isolate Italy 309/04, was shown to have been derived from a live vaccine. Virulence due to the five nucleotide mutations associated with the reversion to virulence was investigated by their addition to the genome of the vaccine strain using reverse genetics. Virulence of these recombinant viruses was determined by infection of 1-day-old turkeys. Disease levels resulting from the combined two matrix mutations was indistinguishable from that produced by the recombinant vaccine, whereas the combined three L gene mutations increased disease to a level (P<0.0001) that was indistinguishable from that caused by the revertant Italy 309/04 virus. Testing of the L mutations individually showed that two mutations did not increase virulence, while the third mutation, corresponding to an asparagine to aspartic acid substitution, produced virulence indistinguishable from that caused by Italy 309/04. In contrast to the vaccine, the virulent mutant also showed increased viability at temperatures typical of turkey core tissues. The notion that increased viral virulence resulted from enhanced ability to replicate in tissues away from the cool respiratory tract, cannot be discounted.