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.

Comparison of enzyme-linked immunosorbent assays and virus neutralization test for detection of antibodies to avian pneumovirus.

Two different whole-virus enzyme-linked immunosorbent assays (ELISAs), developed in Ohio (OH) with APV/Minnesota/turkey/2a/97 and in Minnesota (MN) with APV/Colorado/turkey/97, and the virus neutralization (VN) test were used to test 270 turkey serum samples from 27 Minnesota turkey flocks for avian pneumovirus (APV) antibodies. In addition, 77 turkey serum samples and 128 ostrich serum samples from Ohio were tested. None of the turkey samples from Ohio had antibodies to APV by the VN test and OH ELISA. The ostrich samples were only tested with the VN test and were all negative for antibodies to APV. For the Minnesota serum samples, 107, 115, and 120 were positive by the VN test, the OH ELISA, and the MN ELISA, respectively. The Kappa values of 0.938 and 0.825 showed excellent agreement between the VN test and the OH ELISA and the MN ELISA, respectively, for detection of antibodies to the APV. The OH ELISA and MN ELISA had sensitivities of 1.0 and 0.953, specificities of 0.950 and 0.889, and accuracies of 0.970 and 0.914, respectively. Our results indicate that the 3 methods are sensitive and specific for diagnosis of the APV infection.

Neonatal avian pneumovirus infection in commercial turkeys.

Eleven market turkey flocks developed a respiratory disease characterized by coughing, swollen sinuses and nasal discharge. These symptoms first appeared between 3 and 16 days of age. Avian pneumovirus (APV) RNA was detected by reverse transcriptase (RT)-polymerase chain reaction (PCR) in six of six flocks tested. APV was detected by immunohistochemistry in turbinates of three of three affected flocks tested. Virus isolation attempts were negative. Ten of 11 flocks became seropositive on the APV enzyme-linked immunosorbent assay. Five weeks prior to hatch of these affected market turkeys, several breeder flocks in one geographic area had developed clinical signs and experienced decline in egg production typical of APV infection. In two breeder flocks, acute and convalescent sera indicated APV infection during the period of declining egg production. Attempts to detect APV RNA by RT-PCR from choanal cleft swabs of newly hatched poults were successful. Attempts to isolate the virus from these PCR-positive samples were negative.

Rapid detection of avian pneumovirus in tissue culture by microindirect immunofluorescence test.

An indirect immunofluorescence (IFA) test with a 96-well, flat-bottomed microplate was developed to detect avian pneumovirus (APV) antigen in Vero cell cultures. Samples of nasal turbinates and swabs from infraorbital sinuses and trachea were collected from 4-week-old poults experimentally inoculated with APV. The APV titers by tissue culture IFA staining were compared with that of visual reading of cytopathic effect (CPE). The ability of IFA staining to detect APV antigen correlated well with visualizing CPE. The use of IFA staining of Vero cell cultures allowed detection of APV in substantially less time than the use of visualizing CPE. In addition, the use of IFA allowed specific identification of the virus in cell culture.

Detection of avian pneumovirus in tissues and swab specimens from infected turkeys.

Conventional nested and TaqMan reverse transcription-polymerase chain reaction (RT-PCR) assays for the detection of avian pneumovirus (APV) were evaluated and compared with virus isolation (VI) for sensitivity and specificity. Respiratory tissues and tracheal swabs were collected from experimentally inoculated turkeys between 1 and 21 days postinoculation (DPI) and tested by all detection methods. APV was detected by both RT-PCR procedures as early as 1 DPI and as late as 17 DPI, whereas virus was isolated only between 3 and 7 DPI. Pooled tracheal swab supernatant and dry swabs were excellent specimens for the detection of APV between 3 and 8 DPI. Turbinate and sinus specimens were the most productive samples over the entire collection period. Both RT-PCR assays were rapid and more sensitive than VI for the detection of APV in tissue and swab specimens from infected turkeys. RT-PCR allows for the rapid detection of APV from a variety of respiratory tissues as well as from dry swabs and tracheal swab supernatants. Antibody to APV was detected in 50% of the sampled APV-inoculated birds at 8 and 9 DPI by enzyme-linked immunosorbent assay (ELISA). Early seroconversion (8-10 DPI) allows antibody detection to be used as a screening tool for APV. Rapid and sensitive detection methods are needed for APV, a highly contagious disease affecting U.S. poultry.

Immunohistochemical detection of avian pneumovirus in formalin-fixed tissues.

An immunohistochemical staining technique (IHC) was developed to detect avian pneumovirus (APV) antigen in formalin-fixed, paraffin-embedded tissue sections using streptavidin-biotin immunoperoxidase staining. Samples of nasal turbinates and infraorbital sinuses were collected from 4-week-old poults experimentally inoculated with APV and from older turkeys infected during naturally occurring outbreaks of avian pneumovirus. Tissue was fixed in 10% buffered neutral formalin, embedded in paraffin, sectioned and stained. Inflammatory changes were observed microscopically in the mucosa and submucosa of the nasal turbinates and infraorbital sinuses of both experimentally inoculated poults and naturally infected birds. Viral antigen was detected by IHC in the ciliated epithelial cells of nasal turbinates and infraorbital sinuses.

Development of a highly sensitive and specific enzyme-linked immunosorbent assay based on recombinant matrix protein for detection of avian pneumovirus antibodies.

The matrix (M) protein of avian pneumovirus (APV) was evaluated for its antigenicity and reliability in an enzyme-linked immunosorbent assay (ELISA) for diagnosis of APV infection, a newly emergent disease of turkeys in United States. Sera from APV-infected turkeys consistently contained antibodies to a 30-kDa protein (M protein). An ELISA based on recombinant M protein generated in Escherichia coli was compared with the routine APV ELISA that utilizes inactivated virus as antigen. Of 34 experimentally infected turkeys, 33 (97.1%) were positive by M protein ELISA whereas only 18 (52.9%) were positive by routine APV ELISA 28 days after infection. None of the serum samples from 41 uninfected experimental turkeys were positive by M protein ELISA. Of 184 field sera from turkey flocks suspected of having APV infection, 133 (72.3%) were positive by M protein ELISA whereas only 99 (53.8%) were positive by routine APV ELISA. Twelve serum samples, which were negative by M protein ELISA but positive by routine APV ELISA, were not reactive with either recombinant M protein or denatured purified APV proteins by Western analysis. This indicates that the samples had given false-positive results by routine APV ELISA. The M protein ELISA was over six times more sensitive than virus isolation (11.5%) in detecting infections from samples obtained from birds showing clinical signs of APV infection. Taken together, these results show that ELISA based on recombinant M protein is a highly sensitive and specific test for detecting antibodies to APV.

[Use of antiviral substances in respiratory tract diseases]. / Zastosowanie substancji przeciwwirusowych w chorobach ukladu oddechowego.

Viral infections are an important factor in pathogenesis of symptom aggravation in COPD, which is usually accompanied by immunological system insufficiency, i.e. local immunity. Respiratory syncytial viruses are of vital importance. The paper presents possibilities of using active antiviral substances: amantadine, remantadine, ribavirine, zanamivir preparation. Substances isolated from natural resources have also been mentioned.

Avian pneumovirus infections of turkeys and chickens.

Avian pneumoviruses (APVs) cause major disease and welfare problems in many areas of the world. In turkeys the respiratory disease and the effect on egg laying performance are clearly defined. However, in chickens, the role of APV as a primary pathogen is less clear, although it is widely believed to be one of the factors involved in Swollen Head Syndrome. The mechanisms of virus transmission over large distances are not understood, but wild birds have been implicated. APV has recently been reported in the USA for the first time and the virus isolated was a different type or possibly a different serotype from the APVs found elsewhere. Good biosecurity is crucial for controlling infection and highly effective vaccines are available for prophylaxis. Although different subtypes and possibly different serotypes exist, there is good cross protection between them. Diagnosis is usually based on serology using ELISAs, but the available kits give variable results, interpretation is difficult and improved diagnostic tests are required.

The sensitivity and specificity of a reverse transcription-polymerase chain reaction assay for the avian pneumovirus (Colorado strain).

A reverse transcription-polymerase chain reaction (RT-PCR) assay for the detection of avian pneumovirus (APV), Colorado strain (US/CO), was evaluated for sensitivity and specificity. The single-tube RT-PCR assay utilized primers developed from the matrix (M) gene sequence of the US/CO APV. The RT-PCR amplified the US/CO APV but did not amplify other pneumoviruses, including the avian pneumoviruses subgroups A and B. The RT-PCR was capable of detecting between 10(0.25) mean tissue culture infective dose (TCID50) and 10(-0.44) TCID50 of the US/CO APV. These results have demonstrated that the single-tube RT-PCR assay is a specific and sensitive assay for the detection of US/CO APV.

A modified enzyme-linked immunosorbent assay for the detection of avian pneumovirus antibodies.

Avian pneumovirus (APV) infection of turkeys in Minnesota was first confirmed in March 1997. Serum samples (n = 5,194) from 539 submissions to Minnesota Veterinary Diagnostic Laboratory were tested by a modified enzyme-linked immunosorbent assay (ELISA). Of these, 2,528 (48.7%) samples from 269 submissions were positive and 2,666 (51.3%) samples from 270 submissions were negative for APV antibodies. Most positive samples were from Kandiyohi, Stearns, Morrison, and Meeker counties in Minnesota. In addition, 10 samples from South Dakota were positive. The sensitivity and specificity of the ELISA test with anti-chicken and anti-turkey conjugates were compared by testing field and experimental sera. The ELISA test with anti-turkey conjugate was more sensitive than that with anti-chicken conjugate. The ELISA tests with antigens prepared with APV strains isolated from Colorado and Minnesota were also compared. No difference was detectable. Currently, the Minnesota Veterinary Diagnostic Laboratory uses an antigen prepared from the Colorado isolate of APV and a goat anti-turkey conjugate in the ELISA test.

Detection of antibodies to avian pneumovirus by a micro-indirect immunofluorescent antibody test.

A micro-indirect immunofluorescent antibody (micro-IFA) test with a 96-well, flat-bottomed microplate was developed for measuring avian pneumovirus (APV) antibodies. Two Japanese APV strains (MM-1, 8597/CV94) isolated at different places and times and Vero cells were used for antigen preparation in this test. The test results were compared with those of a serum neutralization (SN) test. By the micro-IFA test, specific immunofluorescent antigens were observed in the cytoplasm of cells infected with either strain, and the antibody titers of antisera to these strains were quite similar. In most cases, the results were obtained within 3 hr. Antibody titers between the micro-IFA and SN tests were highly correlated, with correlation coefficients of 0.873 (MM-1 strain) and 0.889 (8597/CV94 strain). We also investigated APV antibody status in two farms for a period of about 2 yr by the micro-IFA test and revealed that APV infections were repeated within these farms. On the basis of these results, we conclude that our micro-IFA test is useful for routine serologic surveys of APV infections, particularly when a large number of samples are to be treated, because this test was time and labor saving relative to SN tests or conventional IFA tests utilizing embryo tracheal organs or coverslip cell cultures.

A reverse transcription-polymerase chain reaction assay for the detection of avian pneumovirus (Colorado strain).

A reverse transcription-polymerase chain reaction assay was developed for the detection of avian pneumovirus (Colorado strain) (APV-Col). The specific primers were designed from the published sequence of the matrix protein gene of APV-Col. The primers amplified a product of 631 nucleotides from APV-Col. The assay identified only APV-Col and did not react with Newcastle disease virus and infectious bronchitis virus.

Serologic evidence of pneumovirus in Chile.

Chicken and turkey serum samples were obtained and tested in an enzyme-linked immunosorbent assay to establish the presence or absence of antibodies to avian pneumovirus in the commercial poultry population of Chile. A total of 600 serum samples representing 15 different poultry farms of the central region of Chile were considered. These samples were obtained from 52 different flocks and included 220 turkey and 380 chicken sera. The results showed three flocks of market turkeys belonging to the same farm and three flocks of laying chickens of three different farms to be positive to antibody against pneumovirus. These results provide serologic evidence for the presence of pneumovirus in Chilean poultry.

Towards the routine application of nucleic acid technology for avian disease diagnosis.

The use of nucleic acid technology (polymerase chain reaction, probing, restriction fragment analysis and nucleotide sequencing) in the study of avian diseases has largely been confined to fundamental analysis and retrospective studies. More recently these approaches have been applied to diagnosis and what one might call real-time epidemiological studies on chickens and turkeys. At the heart of these approaches is the identification and characterisation of pathogens based on their genetic material, RNA or DNA. Among the objectives has been the detection of pathogens quickly combined with the simultaneous identification of serotype, subtype or genotype. Nucleic acid sequencing also gives a degree of characterisation unmatched by other approaches. In this paper we describe the use of nucleic acid technology for the diagnosis and epidemiology of infectious bronchitis virus, turkey rhinotracheitis virus (avian pneumovirus) and Newcastle disease virus.

Detection of turkey rhinotracheitis virus from chickens with swollen head syndrome by reverse transcriptase-polymerase chain reaction (RT-PCR).

We developed a sensitive and specific method, reverse transcriptase-polymerase chain reaction (RT-PCR) method, for detection of turkey rhinotracheitis virus (TRTV). Two sets of primers were designed from F protein gene sequence of TRTV 3B strain. Sensitivity of detection by nested PCR with the primers corresponded to 0.4 TCID50. Applying this method to a field case of swollen head syndrome (SHS), TRTV could be detected directly from chicken trachea and turbinates. It was identified that this method was very useful to examine the relation of TRTV and SHS.