Turkey Rhinotracheitis Research Articles

Avian Metapneumovirus Subtypes Circulating in Brazilian Vaccinated and Nonvaccinated Chicken and Turkey Farms

Avian metapneumovirus (AMPV) causes turkey rhinotracheitis and is associated with swollen head syndrome in chickens, which is usually accompanied by secondary infections that increase mortality. AMPVs circulating in Brazilian vaccinated and nonvaccinated commercial chicken and turkey farms were detected using a universal reverse transcriptase (RT)-PCR assay that can detect the four recognized subtypes of AMPV. The AMPV status of 228 farms with respiratory and reproductive disturbances was investigated. AMPV was detected in broiler, hen, breeder, and turkey farms from six different geographic regions of Brazil. The detected viruses were subtyped using a nested RT-PCR assay and sequence analysis of the G gene. Only subtypes A and B were detected in both vaccinated and nonvaccinated farms. AMPV-A and AMPV-B were detected in 15 and 23 farms, respectively, while both subtypes were simultaneously found in one hen farm. Both vaccine and field viruses were detected in nonvaccinated farms. In five cases, the detected subtype was different than the vaccine subtype. Field subtype B virus was detected mainly during the final years of the survey period. These viruses showed high molecular similarity (more than 96% nucleotide similarity) among themselves and formed a unique phylogenetic group, suggesting that they may have originated from a common strain. These results demonstrate the cocirculation of subtypes A and B in Brazilian commercial farms.

Development, standardization and assessment of PCR systems for purity testing of avian viral vaccines

The European Pharmacopoeia (Ph. Eur.) requires avian viral vaccines to be free of adventitious agents. Purity testing is an essential quality requirement of immunological veterinary medicinal products (IVMPs) and testing for extraneous agents includes monitoring for many different viruses. Conventional virus detection methods include serology or virus culture, however, molecular tests have become a valid alternative testing method. Nucleic acid testing (NAT) is fast, highly sensitive and has a higher degree of discrimination than conventional approaches. These advantages have led to the development and standardization of polymerase chain reaction (PCR) assays for the detection of avian leucosis virus, avian orthoreovirus, infectious bursal disease virus, infectious bronchitis virus, Newcastle disease virus, infectious laryngotracheitis virus, influenza A virus, Marek's disease virus, turkey rhinotracheitis virus, egg drop syndrome virus, chicken anaemia virus, avian adenovirus and avian encephalomyelitis virus. This paper reviews the development, standardization and assessment of PCR for extraneous agent testing in IVMPs with examples from an Official Medicines Control Laboratory (OMCL).

Viral respiratory diseases (ILT, aMPV infections, IB): are they ever under control?

1. The use of vaccines is the main approach to control of the economically important poultry viral respiratory diseases infectious laryngotracheitis (ILT), avian metapneumovirus (aMPV) infections and infectious bronchitis (IB). This paper appraises the current methods of vaccine control in the light of the nature of each virus and epidemiological factors associated with each disease. 2. Infectious laryngotracheitis virus (ILTV) exists as a single type with a wide range of disease severity. It is a serious disease in certain regions of the world. Recent work has distinguished molecular differences between vaccine and field strains and vaccine virus can be a cause of disease. Vaccines have remained unaltered for many years but new ones are being developed to counter vaccine side effects and reversion and reactivation of latent virus. 3. Avian metapneumoviruses, the cause of turkey rhinotracheitis and respiratory disease in chickens exists as 4 subtypes, A, B, C and D. A and B are widespread and vaccines work well provided that accurate doses are given. Newer vaccine developments are designed to eliminate reversion and possibly counter the appearance of newer field strains which may break through established vaccine coverage. 4. IB presents the biggest problem of the three. Being an unstable RNA virus, part of the viral genome that codes for the S1 spike gene can undergo mutation and recombination so that important antigenic variants can appear irregularly which may evade existing vaccine protection. While conventional vaccines work well against homologous types, new strategies are needed to counter this instability. Molecular approaches involving tailoring viruses to suit field challenges are in progress. However, the simple use of two genetically different vaccines to protect against a wide range of heterologous types is now a widespread practice that is very effective. 5. None of the three diseases described can claim to be satisfactorily controlled and it remains to be seen whether the newer generations of vaccines will be more efficacious and cost effective. The importance of constant surveillance is emphasised and the testing of novel vaccines cannot be achieved without the use of vaccine-challenge experiments in poultry.

Effects of Cyclosporin A induced T-lymphocyte depletion on the course of avian Metapneumovirus (aMPV) infection in turkeys

The avian Metapneumovirus (aMPV) causes an economically important acute respiratory disease in turkeys (turkey rhinotracheitis, TRT). While antibodies were shown to be insufficient for protection against aMPV-infection, the role of T-lymphocytes in the control of aMPV-infection is not clear. In this study we investigated the role of T-lymphocytes in aMPV-pathogenesis in a T-cell-suppression model in turkeys. T-cell-intact turkeys and turkeys partly depleted of functional CD4(+) and CD8(+) T-lymphocytes by Cyclosporin A (CsA) treatment were inoculated with the virulent aMPV subtype A strain BUT 8544. CsA-treatment resulted in a significant reduction of absolute numbers of circulating CD4(+) and CD8alpha(+) T-lymphocytes by up to 82 and 65%, respectively (P<0.05). Proportions of proliferating T-cells within mitogen-stimulated peripheral blood mononuclear cells were reduced by similar levels in CsA-treated birds compared to untreated controls (P<0.05). CsA-treated turkeys showed delayed recovery from aMPV-induced clinical signs and histopathological lesions and a prolonged detection of aMPV in choanal swabs. The results of this study show that T-lymphocytes play an important role in the control of primary aMPV-infection in turkeys.

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.

Production of monoclonal antibodies for Avian Metapneumovirus (SHS-BR-121) isolated in Brazil

Avian Metapneumovirus (aMPV), also called Turkey Rhinotracheitis Virus (TRTV), is an upper respiratory tract infection of turkeys, chickens and other avian species. Five monoclonal antibodies (MAbs) were created against the Brazilian isolate (SHS-BR-121) of aMPV, MAbs 1A5B8; 1C1C4; 2C2E9 and 2A4C3 of IgG1 and MAb 1C1F8 of IgG2a. Four Mabs (1A5B8; 1C1C4; 2C2E9 and 2A4C3) showed neutralizing activity and three (1A5B8; 1C1C4 and 2A4C3) inhibited cellular fusion in vitro. These MAbs were used to investigate antigenic relationship among three strains (SHS-BR-121, STG 854/88 and TRT 1439/91) of aMPV subtypes A and B using cross-neutralization test. The results confirm that the monoclonal antibodies described can be used as a valuable tool in the epizootiological and serological studies, and also for the specific diagnosis of the subtypes in the infection for Avian Metapneumovirus.

Avian and Human Metapneumovirus

Pneumovirus infection remains a significant problem for both human and veterinary medicine. Both avian pneumovirus (aMPV, Turkey rhinotracheitis virus) and human metapneumovirus (hMPV) are pathogens of birds and humans, which are associated with respiratory tract infections. Based on their different genomic organization and low level of nucleotide (nt) and amino acid (aa) identity with paramyxoviruses in the genus Pneumovirus, aMPV and hMPV have been classified into a new genus referred to as Metapneumovirus. The advancement of our understanding of pneumovirus biology and pathogenesis of pneumovirus disease in specific natural hosts can provide us with strategies for vaccine formulations and combined antiviral and immunomodulatory therapies.

Comparison of avian cell substrates for propagating subtype C avian metapneumovirus

Avian metapneumovirus (AMPV) is a respiratory viral pathogen that causes turkey rhinotracheitis (TRT) or swollen head syndrome (SHS) in chickens. AMPV was first isolated in South Africa during the early 1970s and has subsequently spread worldwide during the 1980s to include Europe, Asia, and South America. In 1996, a genetically distinct AMPV subgroup C was isolated in the US following an outbreak of TRT. Vero cells are currently the best available substrate for AMPV propagation but are of non-avian origin. A number of different avian cell substrates have been compared to determine which is the most suitable for the propagation of AMPV to sufficiently high titers. Of the cell substrates tested, primary turkey turbinate and kidney and chicken kidney cells produced titers equal to or greater than Vero cells. Turkey turbinate and kidney epithelial cells that were life-span extended by the ectopic expression of human telomerase catalytic subunit (HTERT) initially displayed AMPV titers comparable to Vero cell controls, but declined in virus production with increased passage in culture. Interestingly, plaques emanating from Vero propagated virus were relatively small and dispersed, when analyzed by immunofluorescent assays (IFA), while both turkey turbinate and kidney cell propagated AMPV produced larger plaques. Even with these differences, there were no changes in the predicted amino acid sequences of the nucleocapsid (N) and phosphoprotein (P) genes of AMPV propagated in either turkey turbinate or Vero host cells. However, the fusion (F) gene showed 11 amino acid differences (98.7% identity) between the two host cell types. These results suggest that AMPV propagated in homologous avian cellular substrates may produce more infectious virus with possibly more effective fusion activity, compared to Vero cell propagation.

Risk Factors Associated with Prevalence of Swollen Head Syndrome (SHS) in Broiler Chickens in Eastern Province - Saudi Arabia

A case-control study was conducted to identify risk factors associated with prevalence of swollen head syndrome (SHS) in broiler chickens in Eastern Province of Saudi Arabia (EPSA). A total of 200 birds - originated from 25 broiler flocks - confirmed cases and 200 controls were used in this study. Source of all data were the necropsy reports of the diagnostic investigations carried out at the Veterinary Teaching Hospital-King Faisal University, between July 1999 and June 2000 and the corresponding results of serological testing for turkey rhinotracheitis virus (TRTV) antibodies. Furthermore, a questionnaire was developed and used to collect data from 47 broiler farms on putative risk factors hypothesized to b e associated with the prevalence of SHS in production farms. Multiple logistic regression was performed to identify factors associated with SHS. Analysis of the data revealed significant association between TRTV antibodies and clinical signs of SHS (Chi =292.6; P<0.001) was concluded. Meanwhile, farm's hygiene and 2

Avian Pneumovirus infection in turkey and broiler farms in Italy: a virological, molecular and serological field survey

Avian Pneumovirus (APV) is the casual agent of Turkey Rhinotracheitis (TRT), and also causes a respiratory infection in chickens, which can result in Swollen Head Syndrome. A survey of APV infection in Italian turkey and broiler farms, in a highly populated area of Northern Italy (Verona Province) is reported. Nine turkey farms and 6 broiler farms were sampled. Sixteen birds from each group were doubly swabbed from the choanal cleft for virus isolation on tracheal organ cultures (TOC) and RT nested PCR (A and B type specific) using extracted RNA. At the same time blood samples were collected for a blocking ELISA serological assay. The broiler samples for virological assays were treated with infectious bronchitis virus (IBV) antiserum raised against serotypes prevalent in the areas sampled, thus avoiding competitive growth of IBV on TOC. Ciliostasis on TOC was taken as the indicator of the presence of the virus, and confirmation was by indirect immunofluorescence. APV was isolated and detected by RT-PCR in 19 day-old turkeys, and in 34, 42 and 48 day-old broilers. All APV strains were found to be type B. All turkeys of more than 4 weeks old were APV positive by ELISA. APV infection was found to be widely spread in the area sampled and the protocol used for virus isolation was shown to be effective. Turkey rhinotracheitis first appeared in Italy in the late 1980s, and the APV isolates involved were subsequently characterised as B types. Our results confirm APV B type to be present in Italy, but the limited findings to date need to be extended by further surveys of other Italian regions.

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.

Characterization of avian metapneumoviruses isolated in the USA

Avian pneumovirus (APV; officially known as turkey rhinotracheitis virus) is an emergent pathogen of birds in the USA that results in upper respiratory tract disease in turkeys. Six years after the first outbreak in the USA, the disease continues to ravage turkey flocks, primarily in the state of Minnesota. From 1997 to 2000, the industry recorded losses estimated at 15 million US dollars per annum. Researchers have developed sensitive diagnostic techniques, including the enzyme-linked immunosorbent assay and the reverse transcriptase-polymerase chain reaction. which, when used together, are highly sensitive in detecting APV outbreaks in commercial turkey flocks. Phylogenetic analysis of the nucleotide and predicted amino acid sequence of 15 US viruses isolated between 1996 and 2000 demonstrated that the US viruses are relatively homogenous but different from the European APV subgroups A and B, resulting in the classification of US isolates into subgroup C. Infectious APV was isolated from sentinel waterfowls placed close to an infected commercial turkey farm and from wild Canada geese captured in Minnesota, suggesting that free-ranging birds may be involved in the spread of APV. Current efforts to prevent and control the infection include improving management and biosecurity practices and developing attenuated live and deletion mutant vaccines capable of conferring protection.

Response of pheasants to live attenuated turkey rhinotracheitis vaccine

The entire crop of 18,120 pheasants for the 2000 rearing season (May 8 to August 7) of one estate in the south of England was vaccinated at one day and...

Occurrence of conjunctivitis, sinusitis and upper region tracheitis in Japanese quail ( Coturnix coturnix japonica ), possibly caused by Mycoplasma gallisepticum accompanied by Cryptosporidium sp. infection

On a farm raising approximately 75 000 Japanese quail ( Coturnix coturnix japonica ) for egg production, the diseased quail showed clinical signs of swelling of the head, nasal discharge, increased lacrimation, and decreased egg production. The flock experienced a mortality rate of 5.7% per day. Macroscopic observation revealed large, gelatinous masses of caseous exudate in the sinuses, egg peritonitis, and airsacculitis. Microscopically, non-purulent or purulent inflammation accompanied by lymphoid hyperplastic tissue with germinal centers was observed in the oculofacial respiratory mucosa. The developing stage of the lesions was abscess formation. In the investigation of pathogens, antigens to Mycoplasma gallisepticum and Pasteurella multocida serotype D were immunolabeled on and demonstrated in the mucosal membranes. In addition, P. multocida , Escherichia coli , Staphylococcus sp., and Streptococcus sp. were isolated from the infraorbital sinuses, and Mycoplasma isolated from a diseased bird was confirmed as M. gallisepticum by polymerase chain reaction (PCR). Furthermore, Cryptosporidium sp. was frequently found in the brush border. Serological, bacteriological and PCR examinations, some with negative outcomes, were carried out concerning microbes that are known to cause swollen heads in birds ( Haemophilus paragallinarum , Newcastle disease virus and turkey rhinotracheitis virus). The average concentration of ammonia fumes in the cages was 30.6 parts/106, which suggests that the high levels of ammonia fumes promoted infection and multiplication of M. gallisepticum in the quail, and that the clinical disease then worsened due to mixed infection with M. gallisepticum and Cryptosporidium sp. or other bacteria.

Detection and differentiation of avian pneumoviruses (metapneumoviruses)

The available detection methods for avian pneumoviruses (turkey rhinotracheitis virus; genus Metapneumovirus ) in turkeys, domestic fowl and other species are reviewed. The advantages and disadvantages of virus isolation techniques, virus or genome (polymerase chain reaction) detection and serology are discussed. Some of the problems likely to be encountered are considered, including the detection of yet to be discovered subtypes, as are the factors that are likely to influence the outcome of the work

Cases of Swollen Head Syndrome in Broiler Chickens in Greece

From 50 commercial broiler flocks included in a study concerning respiratory disease, signs of swollen head syndrome (SHS) were shown in eight. Postmortem examination was performed in eight birds showing signs of SHS from each flock. The trachea and head from each bird were collected for laboratory investigation. An enzyme-linked immunosorbent assay (ELISA) was used for the detection of viral and avian mycoplasma antigens in the trachea, and bacteriologic examinations were performed from the infraorbital sinuses of the infected birds. According to the ELISA results, the most frequently detected antigen in the trachea was Mycoplasma synoviae (six flocks, 75%), followed by infectious bronchitis virus (IBV) (five flocks, 62.5%), avian adenovirus (four flocks, 50%), avian reovirus (three flocks, 37.5%), Mycoplasma gallisepticum (one flock, 12.5%), and Newcastle disease virus (NDV) (one flock, 12.5%). Turkey rhinotracheitis (TRT), infectious laryngotracheitis, and avian influenza viral antigens were not detected. Experimental assays for characterization of NDV and IBV isolates showed that they were strains of low virulence (evidently vaccine strains). Bacteriologic examinations from the infraorbital sinuses of the affected birds resulted in the isolation of Escherichia coli (seven cases, 87.5%) and Staphylococcus spp. (one case, 12.5%). It is evident that TRT virus did not play a causal role in SHS in commercial broiler flocks in Greece, but in this condition, other viruses (IBV, NDV), mycoplasmas, or bacteria may be involved, and environmental conditions seem to be essential to the occurrence and severity of the disease.

PCR-based detection of an emerging avian pneumovirus in US turkey flocks.

Avian pneumovirus (APV) or turkey rhinotracheitis virus (TRTV) is an important respiratory pathogen of domesticated poultry in many countries in Europe, Africa, and Asia. Until recently, the United States was considered free of APV. In late 1996, an atypical upper respiratory tract infection appeared in turkey flocks in Colorado and shortly thereafter in turkey flocks in Minnesota. An avian pneumovirus (APV-US) that was serologically distinct from the previously described TRTV was isolated as the primary cause of the new syndrome. The nucleotide sequence of a fragment of the APV-US fusion gene was determined and used to develop a polymerase chain reaction-based assay that specifically detects APV-US viral nucleic acid sequences in RNA extracts of tracheal swabs and turbinate homogenates. The assay is highly sensitive in that it can detect <0.01 TCID50 of APV. The availability of this assay enables the rapid and accurate determination of APV-US in infected poultry flocks.

Avian pneumovirus infection of laying hens: Experimental studies

Administration of a virulent strain of avian pneumovirus (APV) to specific pathogen free laying hens by the oculonasal route failed to induce a drop in egg production or any adverse effects on eggshell quality. However, intravenous (i.v.) inoculation of the same strain caused a substantial drop in egg production and a high incidence of soft and thin-shelled eggs. Some respiratory signs were also observed and the hens appeared sick, with diarrhoea being observed in approximately one-half of the hens between 4 and 11 days post-inoculation (p.i.). APV antigen was detected in the oviduct epithelium up to 9 days p.i. This challenge model was then used to investigate the efficacy of live attenuated turkey rhinotracheitis (TRT) vaccine administered alone at 1 day old, or an inactivated TRT vaccine (at 16 weeks), or a combined programme using both vaccines, in protecting against this challenge. Neither the live nor the inactivated vaccine alone protected against clinical signs (respiratory infection or diarrhoea). However, the inactivated, but not the live, vaccine did protect against the effect of the i.v. challenge on laying performance. In contrast, the combined vaccination programme protected completely against both clinical signs and poor egg-laying performance. This protection lasted until at least 60 weeks of age. On the basis of the results with this experimental model, it is concluded that the use of live priming followed by administration of inactivated TRT vaccine is necessary to provide complete protection of laying chickens against APV challenge.

Presence of avian pneumovirus type A in continental Europe during the 1980s.

Three isolates of avian pneumovirus (APV) were isolated in Germany during 1987 and 1988 from turkeys with clinical signs of turkey rhinotracheitis and one was isolated during 1990 from a broiler breeder flock with typical signs of swollen head syndrome. The isolates were typed using type-specific reverse transcriptase-polymerase chain reactions. The three isolates from turkeys were identified as type A, while the isolate from the broiler breeders was type B. During the late 1980s no APV live-virus vaccine was used in poultry flocks in Germany, which is indicative of the presence of both types at that time. Previous isolates detected from elsewhere in Europe during the 1980s had been only of type B.

Lack of Antigenic Relationship between French and Recent North American Non-A/Non-B Turkey Rhinotracheitis Viruses

Twelve turkey rhinotracheitis viruses (TRTVs) including the Colorado isolate and two French non-A/non-B viruses were serologically compared. Six enzyme-linked immunosorbent assay (ELISA) antigens derived from subgroup A, subgroup B, a French non-A/non-B, and the Colorado TRTVs were used. Virus neutralization (VN) tests were performed with four Ma-104-adapted viruses derived from subgroup A, subgroup B, a French non-A/non-B, and the Colorado viruses. French strains isolated since 1995 were assigned to subgroup B in both ELISA and VN, whereas those isolated in 1985 and 1986 appeared more diverse: two strains belonged to subgroup B, one to subgroup A, and two others appeared antigenically different from both the A and B subgroups and are classified as non-A/non-B. The Colorado strain appeared different from these three groups of TRTVs. Assignment to subgroup A or B was confirmed by reverse transcription-polymerase chain reaction, but neither the French non-A/non-B strains nor the Colorado virus could be classified with the subgroup-specific G-based primers. These results suggest that at least three antigenically different viruses were present in France in 1985-86 and that the Colorado strain is different from all European TRTVs. Further serologic and phylogenic studies will be necessary to evaluate their actual prevalences and relationships.