Comparative pathogenesis of a subtype A with a subtype B avian pneumovirus in turkeys

This paper describes a study in which the pathogenesis of avian pneumovirus strains, isolated in Belgium, and belonging to the two subtypes A and B, were compared in 2-week-old turkeys. After oculonasal inoculation, animals were either observed for clinical signs or killed for pathological and virological examination. Virus titration and immunofluorescence were performed on the conjunctivae, turbinates, sinuses, upper and lower part of the trachea, lungs and air sacs. No differences were seen between the two subtypes concerning respiratory signs, or macroscopic and microscopic lesions in the respiratory tract. Slight variations were found in site and extent of virus replication. First, only subtype A was able to invade the lower parts of the respiratory tract (bronchi), whereas viral antigens were not detected in the lungs with subtype B. Secondly, the subtype A strain infected two times more epithelial cells at all levels of the upper respiratory tract compared to subtype B. Thirdly, the amount of virus produced at different sites along the respiratory tract was lower in subtype B-inoculated turkeys than in subtype A-inoculated ones.     

Avian metapneumovirus infection of chicken and turkey tracheal organ cultures: comparison of virus–host interactions

Avian metapneumovirus (aMPV) is a pathogen with worldwide distribution, which can cause high economic losses in infected poultry. aMPV mainly causes infection of the upper respiratory tract in both chickens and turkeys, although turkeys seem to be more susceptible. Little is known about virus–host interactions at epithelial surfaces after aMPV infection. Tracheal organ cultures (TOC) are a suitable model to investigate virus–host interaction in the respiratory epithelium. Therefore, we investigated virus replication rates and lesion development in chicken and turkey TOC after infection with a virulent aMPV subtype A strain. Aspects of the innate immune response, such as interferon-α and inducible nitric oxide synthase mRNA expression, as well as virus-induced apoptosis were determined. The aMPV-replication rate was higher in turkey (TTOC) compared to chicken TOC (CTOC) (P?<?0.05), providing circumstantial evidence that indeed turkeys may be more susceptible. The interferon-α response was down-regulated from 2 to 144 hours post infection in both species compared to virus-free controls (P?<?0.05); this was more significant for CTOC than TTOC. Inducible nitric oxide synthase expression was significantly up-regulated in aMPV-A-infected TTOC and CTOC compared to virus-free controls (P?<?0.05). However, the results suggest that NO may play a different role in aMPV pathogenesis between turkeys and chickens as indicated by differences in apoptosis rate and lesion development between species. Overall, our study reveals differences in innate immune response regulation and therefore may explain differences in aMPV – A replication rates between infected TTOC and CTOC, which subsequently lead to more severe clinical signs and a higher rate of secondary infections in turkeys.     

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.     

Subtype B avian metapneumovirus resembles subtype A more closely than subtype C or human metapneumovirus with respect to the phosphoprotein,and second matrix and small hydrophobic proteins

Avian metapneumovirus (aMPV) subtype B (aMPV/B) nucleotide sequences were obtained for the phosphoprotein (P), second matrix protein (M2), and small hydrophobic protein (SH) genes. By comparison with sequences from other metapneumoviruses, aMPV/B was most similar to subtype A aMPV (aMPV/A) relative to the US subtype C isolates (aMPV/C) and human metapneumovirus (hMPV). Strictly conserved residues common to all members of the Pneumovirinae were identified in the predicted amino acid sequences of the P and M2 protein-predicted amino acid sequences. The Cys(3)-His(1) motif, thought to be important for binding zinc, was also present in the aMPV M2 predicted protein sequences. For both the P and M2-1 protein-predicted amino acid sequences, aMPV/B was most similar to aMPV/A (72 and 89% identity, respectively), having only approximately 52 and 70% identity, respectively, relative to aMPV/C and hMPV. Differences were more marked in the M2-2 proteins, subtype B having 64% identity with subtype A but < or = 25% identity with subtype C and hMPV. The A and B subtypes of aMPV had predicted amino acid sequence identities for the SH protein of 47%, and less than 20% with that of hMPV. An SH gene was not detected in the aMPV/C. Phylogenetically, aMPV/B clustered with aMPV/A, while aMPV/C grouped with hMPV.     

Comparison of the efficacy of four antimicrobial treatment schemes against experimental Ornithobacterium rhinotracheale infection in turkey poults pre-infected with avian pneumovirus.

The clinical efficacy of drinking-water administration of enrofloxacin for 3 and 5 days, amoxicillin for 5 days and florfenicol for 5 days for the treatment of respiratory disease induced by an experimental Ornithobacterium rhinotracheale infection in turkeys pre-infected with avian pneumovirus (APV) was assessed based on clinical, bacteriological and histopathological examinations. Experimental groups of 15 susceptible 3-week-old turkeys were each inoculated oculonasally with APV subtype A and 3 days later with susceptible O. rhinotracheale bacteria. Antimicrobial treatment started 1 day after O. rhinotracheale inoculation. After infection, the birds were examined and scored for clinical signs, swabbed daily and weighed at different times. Five birds were euthanized and examined for macroscopic lesions at necropsy at 5 days post bacterial inoculation, and the remainder at 15 days post bacterial inoculation. Samples of the turbinates, trachea, lungs, air sacs, heart and pericardium were collected for bacteriological and/or histological examination. Recovery from respiratory disease caused by an APV/O. rhinotracheale dual infection was most successful after enrofloxacin treatment, irrespective of treatment duration, followed by florfenicol. Amoxicillin treatment was not efficacious. Clinical signs and the number of O. rhinotracheale organisms re-isolated from the trachea and the different respiratory organs were significantly reduced by enrofloxacin treatment for 3 and 5 days. O. rhinotracheale bacteria were not re-isolated from the tracheas of the birds treated with enrofloxacin except for one bird in the 5-day group, as early as 1 day after medication onset. In the group treated with enrofloxacin for 5 days, O. rhinotracheale organisms with a higher minimal inhibitory concentration value (x8) were isolated starting 2 days following treatment onset, initially from a single turkey and subsequently from the other animals.     

Nucleotide and predicted amino acid sequence-based analysis of the avian metapneumovirus type C cell attachment glycoprotein gene: phylogenetic analysis and molecular epidemiology of U.S. pneumoviruses

A serologically distinct avian metapneumovirus (aMPV) was isolated in the United States after an outbreak of turkey rhinotracheitis (TRT) in February 1997. The newly recognized U.S. virus was subsequently demonstrated to be genetically distinct from European subtypes and was designated aMPV serotype C (aMPV/C). We have determined the nucleotide sequence of the gene encoding the cell attachment glycoprotein (G) of aMPV/C (Colorado strain and three Minnesota isolates) and predicted amino acid sequence by sequencing cloned cDNAs synthesized from intracellular RNA of aMPV/C-infected cells. The nucleotide sequence comprised 1,321 nucleotides with only one predicted open reading frame encoding a protein of 435 amino acids, with a predicted M(r) of 48,840. The structural characteristics of the predicted G protein of aMPV/C were similar to those of the human respiratory syncytial virus (hRSV) attachment G protein, including two mucin-like regions (heparin-binding domains) flanking both sides of a CX3C chemokine motif present in a conserved hydrophobic pocket. Comparison of the deduced G-protein amino acid sequence of aMPV/C with those of aMPV serotypes A, B, and D, as well as hRSV revealed overall predicted amino acid sequence identities ranging from 4 to 16.5%, suggesting a distant relationship. However, G-protein sequence identities ranged from 72 to 97% when aMPV/C was compared to other members within the aMPV/C subtype or 21% for the recently identified human MPV (hMPV) G protein. Ratios of nonsynonymous to synonymous nucleotide changes were greater than one in the G gene when comparing the more recent Minnesota isolates to the original Colorado isolate. Epidemiologically, this indicates positive selection among U.S. isolates since the first outbreak of TRT in the United States.     

Synergy between avian pneumovirus and Ornithobacterium rhinotracheale in turkeys

The purpose of this study was to assess the possible synergism between Ornithobacterium rhinotracheale (ORT) and avian pneumovirus (APV), inoculated into turkeys via the natural route, for the reproduction of respiratory disease. Three-week-old specific pathogen free turkeys were inoculated oculonasally with either APV subtype A, ORT or both agents using two different time intervals (3 and 5 days) between APV and ORT. The birds were observed clinically on a daily basis and swabbed intratracheally at short, regular intervals. They were killed at 1, 3, 5, 8 and 15 days post single or dual inoculation and examined for gross lesions at necropsy. Samples of the turbinates, trachea, lungs, air sacs, heart, pericardium and liver were taken for bacteriological and/or histological examination. Combined APV/ORT infections resulted in overt clinical signs and a longer persistence of ORT in the respiratory tract and aggravated the macroscopic and histological lesions in comparison with the groups given single infections. In all ORT-challenged turkeys, ORT was isolated from the turbinates, trachea and lungs, but in turkeys infected with both agents ORT was frequently found in the air sacs and on a single occasion in the heart and pericardium. The time interval between APV and ORT inoculation did not have a significant effect on the outcome of the dual infection. A conspicuous important feature was the attachment of ORT to the cilia of the epithelium of the turbinates and trachea of both ORT-infected and APV/ORT-infected birds. In conclusion, the results show that ORT is able to adhere to and colonize the respiratory tract but, under the circumstances used in this study, is not capable of inducing respiratory disease without viral priming.     

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.     

Compromised T-cell immunity in turkeys may lead to an unpredictable avian metapneumovirus vaccine response and variable protection against challenge

Avian metapneumovirus (aMPV) is an important respiratory pathogen of turkeys with considerable economic impact on poultry production. Although vaccination is widely used for the control of the disease, questions regarding vaccine safety and efficacy remain to be elucidated. This report describes the problems associated with reproducibility of the aMPV-vaccine response, comparing T-lymphocyte-compromised and T-cell-intact turkeys. In three consecutive experiments, turkeys partially depleted of T-lymphocytes by treatment with cyclosporin A as well as untreated turkeys were vaccinated with a commercial live aMPV subtype A (aMPV-A) vaccine at 2 weeks of age. Two weeks later they were challenged with a virulent aMPV-A strain. Despite similar genetic background of the turkeys, comparable housing conditions under isolation and the application of the same aMPV-A vaccine, considerable variation was observed among the experiments regarding replication of the vaccine virus, vaccine-induced clinical signs and protection against challenge infection. The results indicate that differences in the outcome of aMPV-A vaccination may be associated with T-lymphocyte suppression and additionally with an interfering aMPV-B vaccine exposure at the hatchery in two of the experiments. Our study provides possible explanations for the variable protection provided by aMPV vaccines under field conditions.     

Comparison of the full-length genome sequence of avian metapneumovirus subtype C with other paramyxoviruses

We determined the nucleotide (nt) sequence of the small hydrophobic (SH), attachment glycoprotein (G), and RNA polymerase (L) genes, plus the leader and trailer regions of the Colorado strain of Avian metapneumovirus subtype C (aMPV/C) in order to complete the genome sequencing. The complete genome comprised of 13,134 nucleotides, with a 40 nt leader at its 3' end and a 45 nt trailer at its 5' end. The aMPV/C L gene was the largest with 6173 nt and consisting of a single open reading frame encoding a 2005 amino acids (aa) protein. Comparison of the aMPV/C SH, G, and L nt and predicted aa sequences with those of Human metapneumoviruses (hMPV) revealed higher nt and aa sequence identities than the sequence identities between the aMPV subtypes A, B, C, and D, supporting earlier finding that aMPV/C was closer evolutionary to hMPV than the other aMPV subtypes.     

Experimental assessment of the pathogenicity of two avian influenza A H5 viruses in ostrich chicks (Struthio camelus) and chickens.

Virus excretion, immune response, and, for chickens, deaths were recorded in 3-week-old ostriches and chickens inoculated by either the intramuscular or intranasal route with one of two influenza A viruses of subtype H5. One of the viruses, A/turkey/England/50-92/91 (H5N1) (50/92), was highly pathogenic for chickens causing 5/5 deaths by each route of inoculation. The other virus, A/ostrich/Denmark-Q/72420/96 (H5N2) (72420/96), isolated from ostriches in quarantine in Denmark during 1996, was of low pathogenicity for chickens, causing no clinical signs by either route of inoculation. No significant clinical signs were seen in any of the ostriches infected with either of the viruses by either route of infection. Both viruses were recoverable from both species up to 12 days post-infection, and low serological responses were detected in surviving infected ostriches and chickens at 21 days after inoculation.     

Development of a nucleoprotein-based enzyme-linked immunosorbent assay using a synthetic peptide antigen for detection of avian metapneumovirus antibodies in Turkey sera

Avian metapneumoviruses (aMPV) cause an upper respiratory tract disease with low mortality but high morbidity, primarily in commercial turkeys, that can be exacerbated by secondary infections. There are three types of aMPV, of which type C is found only in the United States. The aMPV nucleoprotein (N) amino acid sequences of serotypes A, B, and C were aligned for comparative analysis. On the basis of the predicted antigenicity of consensus sequences, five aMPV-specific N peptides were synthesized for development of a peptide antigen enzyme-linked immunosorbent assay (aMPV N peptide-based ELISA) to detect aMPV-specific antibodies among turkeys. Sera from naturally and experimentally infected turkeys were used to demonstrate the presence of antibodies reactive to the chemically synthesized aMPV N peptides. Subsequently, aMPV N peptide 1, which had the sequence 10-DLSYKHAILKESQYTIKRDV-29, with variations at only three amino acids among aMPV serotypes, was evaluated as a universal aMPV ELISA antigen. Data obtained with the peptide-based ELISA correlated positively with total aMPV viral antigen-based ELISAs, and the peptide ELISA provided higher optical density readings. The results indicated that aMPV N peptide 1 can be used as a universal ELISA antigen to detect antibodies for all aMPV serotypes.     

The pathogenesis of turkey rhinotracheitis virus in turkey poults inoculated with the virus alone or together with two strains of bacteria

Turkey rhinotracheitis virus (TRTV) was recovered in large amounts from the upper respiratory tract of young turkey poults for about 5 days after experimental eyedrop inoculation with either a virulent or an attenuated strain. Thereafter, small amounts of virus were isolated, but only for up to 14 days after inoculation. Virulent TRTV could be recovered infrequently from the internal organs or the intestinal tract but only if bacteria (Bordetella avium and a Pasteurella-like organism) were administered contemporaneously. The results of transmission studies were in agreement with the virus isolation results since they showed that virus could be transmitted from inoculated poults to TRTV-free poults placed in direct contact with them only during the first 9 days after inoculation. Whether inoculated alone or together with TRTV, B. avium colonised the upper respiratory tract to the same degree but apparently did not invade. In contrast, the Pasteurella-like organism did not colonise when inoculated alone but could be recovered from the respiratory tract when administered simultaneously with either a virulent or an attenuated strain of TRTV.     

Development of a Nucleoprotein-Based Enzyme-Linked Immunosorbent Assay Using a Synthetic Peptide Antigen for Detection of Avian Metapneumovirus Antibodies in Turkey Sera

Avian metapneumoviruses (aMPV) cause an upper respiratory tract disease with low mortality but high morbidity, primarily in commercial turkeys, that can be exacerbated by secondary infections. There are three types of aMPV, of which type C is found only in the United States. The aMPV nucleoprotein (N) amino acid sequences of serotypes A, B, and C were aligned for comparative analysis. On the basis of the predicted antigenicity of consensus sequences, five aMPV-specific N peptides were synthesized for development of a peptide antigen enzyme-linked immunosorbent assay (aMPV N peptide-based ELISA) to detect aMPV-specific antibodies among turkeys. Sera from naturally and experimentally infected turkeys were used to demonstrate the presence of antibodies reactive to the chemically synthesized aMPV N peptides. Subsequently, aMPV N peptide 1, which had the sequence 10-DLSYKHAILKESQYTIKRDV-29, with variations at only three amino acids among aMPV serotypes, was evaluated as a universal aMPV ELISA antigen. Data obtained with the peptide-based ELISA correlated positively with total aMPV viral antigen-based ELISAs, and the peptide ELISA provided higher optical density readings. The results indicated that aMPV N peptide 1 can be used as a universal ELISA antigen to detect antibodies for all aMPV serotypes.