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.     

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.     

A single subtype of avian pneumovirus circulates among Minnesota turkey flocks.

The recent emergence of avian pneumovirus (APV) infection among US turkey flocks has resulted in a major economic threat to the turkey industry. In order to elucidate the molecular epidemiology of APV, comparative sequence analysis of the fusion (F) protein gene of APV was performed for 3 cell culture-adapted isolates and 10 APV positive clinical samples recovered from US turkey flocks. Relatively modest levels of nucleotide and amino acid sequence divergence were identified, suggesting the prevalence of a single lineage of APV among US turkey flocks. Additionally, numerous polymorphisms were identified that were only represented in the clinical samples but not in the in vitro propagated isolates of APV. Phylogenetic analyses confirm that the subtype of APV circulating in the upper Midwestern United States is evolutionarily related to, but distinct from, European APV subgroups A and B. Overall, the results of the present investigation suggest that there has been only a single recent introduction of APV into US turkey populations in the upper Midwestern United States.     

Enteric viruses in diarrheic turkey poults

Thirty-three intestinal samples from 10-to-21-day-old diarrheic turkey poults were examined for the presence of enteric viruses by electron microscopy. Samples originated from 32 flocks in six commercial operations located in six states. Mortality in these flocks ranged from 3 to 15%, and birds from recovered flocks varied greatly in size. Rotavirus-like agents (RVLA) were the most common viruses associated with diarrhea outbreaks in the flocks examined, occurring in five out of six operations. Other viruses detected either singly or in combination, in order of prevalence, were astroviruses, reoviruses, rotaviruses, enteroviruses, and adenoviruses. With the exception of RVLA and rotaviruses, the other viruses were identified solely on the basis of morphology. Salmonellae were isolated from only one of the intestinal samples. By electron microscopy, RVLA were morphologically indistinguishable from rotaviruses, occurring as both 55-nm single-shelled and 70-nm double-shelled particles. However, immune electron microscopy was useful for antigenic differentiation of these two viruses. Turkey rotaviruses reacted with antisera to porcine and bovine rotaviruses, whereas turkey RVLA did not. Neither turkey rotaviruses nor RVLA reacted with antisera to porcine para-rotavirus or an antigenically distinct bovine rotavirus (bovine rotavirus-like agent). Similarly, convalescent anti-turkey RVLA serum (from recovered specific-pathogen-free poults) reacted with homologous virus but did not react with mammalian or avian rotaviruses or reoviruses. Further, RVLA were found to possess RNA electrophoretic migration patterns unlike those of conventional rotaviruses or reoviruses. This trait was used as an additional means of differentiating these viruses.     

Characteristics of fowl cholera outbreaks in turkeys in Georgia in 1986

Information was gathered from 64 cases of fowl cholera (FC) in turkey flocks through diagnostic case records, flock records, and telephone and mail surveys. Forty-five cases came from flocks of commercial turkeys, of which 15 were presented twice, and four came from mature breeder flocks. The prevalence of FC was 18.0% of commercial flocks and 14.7% of breeder flocks at risk. The average age at first diagnosis of FC was 90 days in commercial turkey flocks and 32 weeks 5 days in breeder flocks. Acute mortality was the most common presenting complaint, with a 0.37% average mortality in commercial flocks on the day of first presentation, 0.80% in commercial flocks presented a second time, and 0.43% in breeder flocks. Pasteurella multocida was cultured from 69.8% of the 361 tissue samples submitted from these cases. Novobiocin, penicillin, and chlortetracycline (CTC) had the greatest in vitro activity against isolates. Serotype 3-cross-4 was found in all 18 commercial flocks from which isolates were typed. All breeder flocks and 88.6% of commercial flocks were vaccinated before disease onset. Flocks were treated for an average of 14.3 days, most commonly with high levels of sulfadimethoxine and/or CTC. Body weights of affected birds were comparable to those of birds in unaffected flocks, but mortality and feed efficiency were worse.     

Risk factors for foot-pad dermatitis in chicken and turkey broilers in France

A prospective cohort study was conducted in the Brittany region to identify the risk factors related to foot-pad dermatitis (considered an indicator of animal well-being) in chicken and turkey broilers reared under commercial conditions. Factors related to the shed, equipment, litter management and stocking density were recorded; the dependent variable was the prevalence of lesions observed on the slaughterhouse chain. Lesions were scored from 0 (no lesion) to 3 (severe lesion). Our survey lasted from May 1999 to October 2000. Fifty flocks of chicken broilers (15 farms), 27 flocks of female turkey broilers (21 farms) and 41 flocks of male turkey broilers (27 farms) were surveyed. In chicken broilers, 10% of flocks were of high quality (80% of birds with score 0) and this was related to the use of concrete floors with a thin layer of wood shavings. In turkey broilers, 48% of female and 46% of male flocks were of bad quality (>10% of birds with score 3). A poor fan ventilation system (<150 m³/h/kg) was a significant risk factor. Turkey flocks of high quality were not observed. Stocking density had no influence on the prevalence of foot-pad dermatitis. We concluded that it is possible under high commercial stocking densities to have flocks with a low prevalence of foot-pad dermatitis in chicken broilers, whereas it is not in turkey broilers. Hence in chicken broilers, implementing a monitoring system based on the observation of foot-pad dermatitis prevalence at slaughter appears to be more appropriate than to legislate stocking density. In turkey broilers, it would probably be necessary either to reduce the stocking density drastically or to investigate new systems of floor drainage.     

Effect of an immunomodulator on the efficacy of an attenuated vaccine against avian pneumovirus in turkeys

Since 1997, avian pneumovirus (APV) has caused estimated annual losses of $15 million to the Minnesota turkey industry. In order to develop an attenuated live vaccine against APV, we serially passaged a Minnesota isolate of APV (APV/MN/turkey/1-a/97) in vitro in cell cultures for 41 passages. Laboratory experiments with this high-passage virus (P41) indicated that the attenuated virus provided immunogenic protection to turkeys against challenge with virulent APV, although some birds showed mild to moderate dinical signs after inoculation. To reduce the residual pathogenicity of P41, while maintaining its immunogenicity, we decided to vaccinate turkeys with P41 in the presence of an immunomodulator, S-28828 (1-n-butyl-2-ethoxymethyl-1H-imidazo[4,5-c]quinolin-4-amine-hydrochloride), which is a potent cytokine inducer. The combined inoculation of S-28828 (5 mg/kg body weight) and P41 resulted in a significant reduction in the incidence of virus-induced clinical signs in comparison with birds that received P41 without immunomodulator (P < 0.05). Only 17% of birds inoculated with S-28828 + APV P41 showed mild respiratory symptoms at 5 days postinoculation as compared with 46% of the vaccinated turkeys that did not receive S-28828. Vaccination with either P41 or with P41 + S-28828 protected turkeys against dinical signs and viral replication after challenge with virulent APV. These results indicate that immunomodulators, such as S-28828, may act as good vaccine adjuvants that can reduce the pathogenicity but maintain the immunogenicity of partially attenuated vaccines.     

Serologic evidence of infectious bursal disease virus infection in Iowa turkeys

Two infectious bursal disease viruses (IBDVs)--a cell-culture-adapted chicken strain designated Edgar strain and a recent isolate from turkeys in Missouri--were used to assay sera from 10 Iowa turkey flocks collected between 1 and 16 weeks of age. The two viruses were serologically distinct in cross-neutralization tests. For all flocks, a similar serologic pattern was found consisting of (1) low maternal antibody titers to turkey IBDV and occasionally to Edgar strain IBDV between 1 and 3 weeks of age, (2) a period of very low or no detectable titers between 3 and 7 weeks of age, and (3) sharply rising high titers to turkey IBDV with low titers to Edgar IBDV beginning at 5 to 8 weeks of age. These findings indicate that infection with IBDV of the serotype represented in this study by the turkey isolate is common in Iowa turkey flocks, whereas infection with IBDV represented by Edgar strain is uncommon. Infection occurred between 3 and 7 weeks of age during the late brooding period or after birds had been moved to an intermediate growing facility. All flocks developed complicated respiratory disease with excessive mortality caused by Escherichia coli septicemia, typically between 3 and 6 weeks of age. Although there was a temporal relationship between IBDV infection and respiratory disease, the possible role of IBDV in the process is unknown.     

Serologic evidence of infectious bursal disease virus serotype II infection in Minnesota turkeys

Serum samples from seven randomly selected Minnesota turkey flocks were tested for antibodies to infectious bursal disease virus serotype I (Lukert strain, isolated from chickens, and North Carolina strain, isolated from turkeys) using a virus-neutralization (VN) test. All flocks were found to have low antibody titers to both Lukert and North Carolina strains. Five out of the seven flocks had high VN titers to the Missouri strain, a serotype II virus isolated from turkeys.     

Clinical signs and mortality caused by Ornithobacterium rhinotracheale in turkey flocks

Upper respiratory tract disease (manifesting itself in rhinitis, tracheitis and conjunctivitis) and mortality associated with Ornithobacterium rhinotracheale infection were observed in four flocks of 2- to 3-week-old turkeys. In a 15- to 16-week-old turkey flock bilateral catarrhal-croupous pneumonia was found in the dead birds. In a further 5-week-old flock and in three 16- to 20-week-old turkey flocks mortality was preceded by nervous signs (motor disturbances, recumbency, abnormal carriage of the head) and was found to be associated with fibrinopurulent inflammation of the cranial bones and meningitis. The bacterium O. rhinotracheale was isolated from the affected organs in the different disease conditions. The isolated strains did not differ markedly in cultural, morphological and biochemical properties. This is the first report of a turkey disease manifesting itself in nervous signs associated with O. rhinotracheale infection.     

Trials with Marek's disease vaccines prepared from a turkey herpes virus and an attenuated Marek's disease virus.

In field trials involving over 224,000 fowls in 11 different commercial flocks, three vaccines were used, namely a freeze-dried vaccine prepared from a turkey herpes virus, a cell-associated virus vaccine prepared from the same isolate and a cell-associated vaccine prepared from a strain of Marek's disease virus isolated from a fowl. The mortality from Marek's disease was reduced by 80 per cent to 95 per cent in birds vaccinated with the freeze-dried vaccine. Cell associated vaccines gave slightly less protection.     

Infection of turkeys with Ornithobacterium rhinotracheale and Mycoplasma synoviae

Within 1 mo, two separate outbreaks of respiratory disease occurred in two flocks on the multiage market turkey farm in Slovenia. More severe dinical signs and higher mortality were observed in male birds. Ornithobacterium rhinotracheale (ORT) was isolated in pure culture from tracheas of the affected birds in both outbreaks. Commercial enzyme-linked immunosorbent assay test showed the presence of antibodies to ORT in sera of birds from both clinically affected flocks and also in two flocks of younger birds without clinical sings. Immunoblotting with ORT culture isolated during the outbreak as an antigen confirmed the presence of antibodies to ORT in sera of turkeys of all four flocks examined. In addition, three different serologic assays also detected antibodies to Mycoplasma synoviae (MS) in three out of four flocks. The concomitant infection with MS did not show an obvious effect on mortality rates nor on the antibody response against ORT. Younger birds appeared to be less susceptible to ORT pathogenicity because in those flocks the infection was subclinical.     

Antigenic cross-reactivity among avian pneumoviruses of subgroups A,B, and C at the matrix but not nucleocapsid proteins

Earlier findings from our laboratory based on analysis of nucleotide and predicted amino acid sequence identities of 15 avian pneumoviruses (APVs) isolated from the United States (subgroup C) demonstrated that the viruses were phylogenetically separated from the European subgroup A and subgroup B viruses. Here, we investigated whether viruses from the three subgroups were cross-reactive by testing field sera positive for each of the APV subgroups in an enzyme-linked immunosorbent assay (ELISA) test with recombinant matrix (M) and nucleoprotein (N) proteins generated from a Minnesota APV isolate (APV/MN2A). Sera from turkeys infected with APV subgroup A, B, or C reacted with recombinant M protein derived from APV/MN2A. In contrast, recombinant N protein from APV/MN2A virus was reactive with sera from subtypes A and C viruses but not from subtype B virus. The results illustrate that viruses from the three APV subtypes share antigenic homology, and the M protein-based ELISA is adequate for monitoring APV outbreaks but not for distinguishing between different subtypes.