Seroprevalence of avian pneumovirus in Minnesota turkeys

Avian pneumovirus (APV) causes respiratory tract infection in turkeys and was first seen in the United States in Colorado in late 1996. In early 1997, the disease was recognized in Minnesota and caused estimated losses of up to 15 million dollars per year. This virus has not been reported in the other turkey producing states. We here report the seroprevalence of APV in Minnesota from August 1998 to July 2002. The average rate of seroprevalence has been 36.3% (range = 14.2%-64.8%). A seasonal bias was observed, with peak incidences in the fall and spring. A higher rate of seropositivity was observed in counties with the highest concentration of turkeys.     

Pathogenic and immunosuppressive effects of avian pneumovirus in turkeys

Avian pneumovirus (APV) causes a respiratory disease in turkeys. The virus has been associated with morbidity and mortality due to secondary infections. Our objective was to determine if APV caused immunosuppression in the T-cell or B-cell compartments and to study the pathogenesis of the disease in APV maternal antibody-lacking 2-wk-old commercial turkeys. APV was administered by the eyedrop/intranasal route. Observations were made for gross lesions, viral genome, and T-cell mitogenesis and cytokine secretion at 3, 5, 7, 14, and 21 days postinoculation (DPI). During the acute phase of the disease that lasted for about 1 wk, the turkeys exposed to APV showed clinical signs characterized by nasal discharge and sinus swelling. Virus genome was detected by in situ hybridization in cells of turbinates and trachea at 3 and 5 DPI. At 3 and 5 DPI, spleen cells of the birds infected with APV markedly decreased proliferative response to concanavalin A (Con A). Con A and lipopolysaccharide stimulation of spleen cells from virus-exposed turkeys resulted in accumulation of nitric oxide-inducing factors (NOIF) in the culture fluid. NOIF were not detected in culture fluids of Con A-stimulated spleen cells of virus-free turkeys. APV did not compromise the antibody-producing ability of turkeys against several extraneous antigens such as Brucella abortus and tetanus toxoid.     

Vaccination of turkeys with an avian pneumovirus isolate from the United States

Four-week-old poults obtained from avian pneumovirus (APV) antibody-free parents were vaccinated with different serial 10-fold dilutions of cell culture-propagated APV vaccine. The birds were vaccinated with 50 microl into each conjunctival space and nostril (total of 200 microl). Each poult of each group was vaccinated in groups that received doses of 4 x 10(4), 4 x 10(3), 4 x 10(2), 4 x 10(1), or 4 x 10(0) 50% tissue culture infective dose (TCID50) of APV vaccine, respectively. Respiratory signs were seen between 3 and 12 days postvaccination (PV) in the poults that were vaccinated with 4 x 10(4), 4 x 10(3), and 4 x 10(2) TCID50, respectively. In these groups, APV was detected from swabs collected at 5 days PV and seroconversion was detected at 2 wk PV. The groups that were originally vaccinated with 4 x 10(1) and 4 x 10(0) TCID50 developed mild clinical signs after vaccination, but neither virus nor antibody was detected PV. At 2 wk PV (6 wk of age), birds from each group, along with five unvaccinated controls, were challenged with APV. Upon challenge, the 4 x 10(4) and 4 x 10(3) TCID50 groups were protected against development of clinical signs and were resistant to reinfection. The group previously vaccinated with 4 x 10(2) TCID50 developed clinical signs after challenge that were considerably milder than those seen in the groups that had previously been vaccinated with lower doses or no virus. Even though 4 x 10(2) TCID50 vaccine dose administered by intranasal ocular route resulted in infection, incomplete protection resulted with this pivotal dose. Upon challenge, the 4 x 10(1) and 4 x 10(0) TCID50 groups exhibited milder disease signs than those seen in the challenged unvaccinated controls. In these groups, APV was detected in preparations of swabs collected at 5 days postchallenge (PC) and seroconversion was detected at 2 wk PC. These results indicate that the dose of APV vaccine that causes protection is higher than that required to produce infection.     

Pathogenic interactions between Chlamydophila psittaci and avian pneumovirus infections in turkeys

Both Chlamydophila psittaci and avian pneumovirus (APV) are highly prevalent in Belgian turkeys and might contribute to the respiratory disease complex observed in turkeys. Initial outbreaks of chlamydiosis occur mostly at the age of 4-8 weeks, often accompanied by an APV infection in APV non-vaccinated farms. Regardless APV vaccination, breakthroughs of APV infection from 8 weeks on do occur, a period when also a second C. psittaci infection appears. Therefore, this study examined the pathogenicity of an APV superinfection in C. psittaci predisposed turkeys. Turkeys were infected with C. psittaci, APV or with C. psittaci followed by APV. Simulating the impact of an APV infection during the acute phase or latent phase of a C. psittaci infection, turkeys have been infected with APV at 1 and 5 weeks post C. psittaci infection, respectively. APV infection during the acute phase of a C. psittaci infection aggravates the severity of clinical signs, macroscopic lesions, pharyngeal APV excretion and histological tracheae lesions. In contrast, no clear interaction could be established after APV infection in latently C. psittaci infected specific pathogen-free (SPF) turkeys. This study clearly demonstrates the exacerbating role of APV during acute C. psittaci infection, which can play an important role in the respiratory disease complex of turkeys.     

Susceptibility of an avian pneumovirus isolated from Minnesota turkeys to physical and chemical agents

Survival characteristics of a Minnesota avian pneumovirus (APV) isolated from a turkey nasoturbinate, propagated in tissue culture, and exposed to various physiochemical treatments were determined. These characteristics included survivability under various conditions. Specifically, APV was viable at temperatures of -70 C and -20 C for over 26 wk, 4 C for less than 12 wk, 20 C for less than 4 wk, 37 C for 48 hr, and 50 C for less than 6 hr. In addition, APV survived 12 freeze/thaw cycles with no loss of activity. With a variable pH for 1 hr, the titer was unaffected between the levels of pH 5 and 9. Several disinfectants, including quaternary ammonia, ethanol, iodophor, a phenol derivative, a biguanide, and bleach, were all effective in reducing the viability of the virus. After 7 days of drying at room temperature, the APV remained viable and was recovered on cell culture.     

Duration of cross-protection between subtypes A and B avian pneumovirus in turkeys

The degree and duration of clinical and virological cross-protection between avian pneumovirus subtypes A and B were examined in two-week-old pneumovirus antibody-free turkeys. The turkeys were inoculated with either a virulent subtype A (Belgian isolate A/T6/96), a virulent subtype B (Belgian isolate B/T9/96), an attenuated subtype A or an attenuated subtype B, and challenged homologously and heterologously with virulent avian pneumovirus two, five and 11 weeks after inoculation. Birds inoculated with virulent A or B virus showed typical respiratory signs from three to seven days after inoculation. After challenge, no clinical signs were observed in any of the groups, and no virus was isolated from the turkeys that had been initially inoculated with a virulent strain. Virulent virus was recovered from the birds that had been initially inoculated with attenuated subtypes and challenged five and/or 11 weeks later with a heterologous virulent strain. Birds challenged after five weeks showed a serological booster reaction only when they had been inoculated initially with a virulent or attenuated subtype B and challenged with subtype A. Seroconversion was observed in all the groups challenged after 11 weeks except when they had been inoculated initially with attenuated subtype B and challenged with subtype B.     

Efficacy of avian pneumovirus vaccines against an avian pneumovirus/Escherichia coli O2:K1 dual infection in turkeys

The clinical, pathological and microbiological outcome of a challenge with avian pneumovirus (APV) and Escherichia coli O2:K1 was evaluated in turkeys vaccinated with an attenuated APV vaccine and with or without maternally derived antibodies. Two groups of two-week-old poults, one with and one without maternally derived antibodies against APV, were vaccinated oculonasally with attenuated APV subtype A or B. A third group remained unvaccinated. Eleven weeks later, the turkeys were inoculated intranasally with either virulent APV subtype A, or E. coli O2:K1, or with both agents three days apart. After the dual infection, birds vaccinated with attenuated subtype A or B, and with or without maternally derived antibodies, had lower mean clinical scores than the unvaccinated birds. In the vaccinated birds, virus replication was significantly reduced and no bacteria were isolated, except from the birds vaccinated with attenuated subtype B. In the unvaccinated turkeys, large numbers of E. coli O2:K1 were isolated from the turbinates of the dually infected birds between one-and-a-half and seven days after they were inoculated.     

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.     

Reduced efficacy of hemorrhagic enteritis virus vaccine in turkeys exposed to avian pneumovirus

Avian pneumovirus (APV) is an immunosuppressive respiratory pathogen of turkeys. We examined the effect of APV infection on the vaccine efficacy of hemorrhagic enteritis virus (HEV) vaccines. APV was inoculated in 2-wk-old turkeys. Two or four days later, an attenuated HEV vaccine (HEVp30) or marble spleen disease virus (MSDV) vaccine were administered. Virulent HEV challenge was given 19 days after HEV vaccination. APV exposure compromised the ability of HEVp30 and MSDV to protect turkeys against virulent HEV. The protective index values were as follows: MSDV (100%) versus APV + MSDV (0%) (P < 0.05); HEVp30 (60%) versus APV + HEVp30 (30%) (P < 0.05) (Experiment I) and HEVp30 (56%) versus APV + HEVp30 (20%) (P < 0.05) (Experiment II). These data indicated that APV reduced the efficacy of HEV vaccines in turkeys.     

Effects of bacterial coinfection on the pathogenesis of avian pneumovirus infection in turkeys

Four- and nine-week-old poults were inoculated with cell culture propagated avian pneumovirus (APV) into each conjunctival space and nostril, followed by inoculation 3 days later with Escherichia coli, Bordetella avium (BA), or Ornithobacterium rhinotracheale or a mixture of all three (EBO). Clinical signs were evaluated on days 3, 5, 7, 9, 11, and 14 postinoculation (PI) of APV. The poults were euthanatized on days 2, 4, 6, 10, and 14 PI, and blood and tissues were collected. The poults that received APV followed by EBO or BA alone developed more severe clinical signs related to nasal discharge and swelling of intraorbital sinuses than did poults inoculated with APV alone or bacteria alone. More severe pathologic changes were found in poults inoculated with APV+BA that extended to the air sacs and lungs, particularly in 9-wk-old poults. Bordetella avium was recovered from tracheas and lungs of birds that were inoculated with APV followed by EBO or BA alone. APV was detected by immunohistochemical staining in the upper respiratory tract longer in the groups of poults inoculated with APV and pathogenic bacteria than in those that received only APV, particularly when BA was involved. Viral antigen was also detected in the lungs of poults that were inoculated with APV followed by administration of EBO or BA alone. Loss of cilia on the epithelial surface of the upper respiratory tract was associated with BA infection and may enhance infection with APV, allowing deeper penetration of the virus into the respiratory tract.     

The clinical, pathological and microbiological outcome of an Escherichia coli O2:K1 infection in avian pneumovirus infected turkeys

The purpose of this study was to evaluate the effect of an Escherichia coli infection in avian pneumovirus (APV)-infected turkeys. One group of 2-week-old specific pathogen-free (SPF) and two groups of 3-week-old conventional (CON) turkeys were inoculated oculonasally with virulent APV subtype A alone, with E. coli O2:K1 alone or with both agents at varying intervals (1, 3, 5 or 7 days) between the two inoculations. The birds were followed clinically and examined for macroscopic lesions at necropsy. Titres of APV were determined in the turbinates, trachea, lungs and air sacs. The number of E. coli O2:K1were assessed in the turbinates, trachea, lungs, air sacs, liver and heart. In both SPF and CON turkeys, dual infection resulted in an increased morbidity and a higher incidence of gross lesions compared to the groups given single infections, especially with a time interval between APV and E. coli inoculations of 3 and 5 days. APV was isolated from the respiratory tract of all APV-infected groups between 3 and 7 days post inoculation. E. coli O2:K1 was isolated only from turkeys that received a dual infection. It was recovered from the turbinates, trachea, lungs, heart and liver. These results show that APV may act as a primary agent predisposing to E. coli colonization and invasion.     

Experimental infection of turkeys with avian pneumovirus and either Newcastle disease virus or Escherichia coli

Avian pneumoviruses (APVs) are RNA viruses responsible for upper respiratory disease in poultry. Experimental infections are typically less severe than those observed in field cases. Previous studies with APV and Escherichia coli suggest this discrepancy is due to secondary agents. Field observations indicate APV infections are more severe with concurrent infection by Newcastle disease virus (NDV). In the current study, we examined the role of lentogenic NDV in the APV disease process. Two-week-old commercial turkey poults were infected with the Colorado strain of APV. Three days later, these poults received an additional inoculation of either NDV or E. coli. Dual infection of APV with either NDV or E. coli resulted in increased morbidity rates, with poults receiving APV/NDV having the highest morbidity rates and displaying lesions of swollen infraorbital sinuses. These lesions were not present in the single APV, NDV, or E coli groups. These results demonstrate that coinfection with APV and NDV can result in clinical signs and lesions similar to those in field outbreaks of APV.     

Detection of mycoplasmas in eye swab specimens of cattle

In the present investigation 262 conjunctival swabs were taken from 178 cattle and examined for mycoplasmas. The isolation was possible from 111 swabs. Mycoplasmas were found in eyes with clinical symptoms of IBK (in 64 of 148 swabs investigated = 43.2%) as well as in healthy eyes (in 47 of 114 swabs investigated = 41.2%). Consequently a correlation between clinical findings and isolation of mycoplasmas could not be observed. Unfortunately 60 of 111 isolates could not be subcultivated after storage at -20 degrees C. Using the indirect immunofluorescence test 41 of the 42 surviving isolates were identified as M. bovoculi which before has not been isolated in the Federal Republic of Germany. One isolate was determined as A. laidlawii. The 17 M. bovoculi strains investigated for their biochemical reactions showed the same characteristics like the reference strain M. bovoculi M 165/69. In repeating examinations mycoplasmas could be isolated 5 times after one month and 14 times after 6 months. Cattle younger than 2 years were more often infected with mycoplasmas (62.5%) than older animals (19.4%). No difference, however, could be observed in the clinical manifestations of IBK between younger and older animals. Mycoplasmas were more frequently isolated in autumn (43.6%) than in spring (21.4%) and summer (29.3%). In most of the animals examined both eyes were colonized by mycoplasmas. No spiroplasmas could be detected in the 262 conjunctival swabs investigated.     

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.     

Cold-adapted strain of avian pneumovirus as a vaccine in one-day-old turkeys and the effect of inoculation routes

To determine the optimum route of vaccination, we inoculated 1-day-old turkeys with a cold-adapted strain of avian pneumovirus (APV) by oculonasal, oral, or aerosol route. Another two groups served as nonvaccinated-challenged and nonvaccinated-nonchallenged groups. Birds in all vaccinated and nonvaccinated-challenged groups were challenged with virulent APV 3 wk postvaccination. After challenge, no vaccinated bird developed clinical signs or virus shedding, whereas nonvaccinated-challenged birds developed clinical signs (clinical score = 11.2/bird) and shed virus from their choanal cleft. Birds in all three vaccinated groups seroconverted at 3 wk postvaccination. The nonvaccinated-nonchallenged group remained free of clinical signs and virus shedding and did not develop APV antibodies throughout the course of the study. These results suggest that this cold-adapted strain of APV is safe and effective in 1-day-old turkeys when given by any of the three routes.     

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.