Pathogenic and immunogenic responses in turkeys following in ovo exposure to avian metapneumovirus subtype C

Commercial turkey eggs, free of antibodies to avian metapneumovirus subtype C (aMPV/C), were inoculated with aMPV/C at embryonation day (ED) 24. There was no detectable effect of virus inoculation on the hatchability of eggs. At 4 days post inoculation (DPI) (the day of hatch (ED 28)) and 9 DPI (5 days after hatch), virus replication was detected by quantitative RT-PCR in the turbinate, trachea and lung but not in the thymus or spleen. Mild histological lesions characterized by lymphoid cell infiltration were evident in the turbinate mucosa. Virus exposure inhibited the mitogenic response of splenocytes and thymocytes and upregulated gene expression of IFN-γ and IL-10 in the turbinate tissue. Turkeys hatching from virus-exposed eggs had aMPV/C-specific IgG in the serum and the lachrymal fluid. At 3 week of age, in ovo immunized turkeys were protected against a challenge with pathogenic aMPV/C.     

B-cell infiltration in the respiratory mucosa of turkeys exposed to subtype C avian metapneumovirus

Turkeys exposed to avian metapneumovirus (aMPV) subtype C showed extensive lymphoid cell infiltrations in the nasal turbinates of the upper respiratory tract. The cellular infiltration occurred after the first virus exposure but not after re-exposure. Quantitation of the relative proportions of mucosal immunoglobulin (Ig)A+, IgG+, and IgM+ cells in controls and virus-exposed turkeys revealed that at 7 days after the first virus exposure, when mucosal infiltration was well pronounced, there was a significant increase (P < 0.05) in the numbers of infiltrating IgA+ but not of IgG+ and IgM+ cells. After the second virus exposure, although the overall numbers of mucosal lymphoid cells were similar in the virus-exposed and control turkeys, the relative proportions of IgA+ and IgG+ cells were significantly higher in the virus-exposed turkeys (P < 0.05) than in controls. Furthermore, elevated levels of aMPV-specific IgA were detected in the nasal secretions and the bile of virus-exposed birds after the second but not after the first virus exposure. These results suggest, for the first time, the possible involvement of local mucosal immunoglobulins in the pathogenesis of aMPV in turkeys.     

Development of a virosome vaccine against avian metapneumovirus subtype C for protection in turkeys

An avian metapneumovirus (aMPV) virosome vaccine was prepared and tested for protection of turkeys by aMPV challenge. The vaccine was produced using a detergent-based (Triton X-100) extraction of aMPV subtype C followed by detergent removal with SM2 Bio-Beads. Western blot and virus-neutralization analysis confirmed that the aMPV virosomes contained both the fusion and attachment glycoproteins. Specific-pathogen-free turkeys were immunized either intranasally (i.n.) or intramuscularly (i.m.) with two doses of the aMPV virosome vaccine. Vaccination decreased clinical signs of disease following virulent challenge, and IN vaccination was superior to i.m. vaccination in reducing clinical signs. Decreases in viral load in the respiratory tract were observed in turkeys receiving i.n. vaccination with aMPV virosomes compared to unvaccinated poults. Increased virus-neutralizing antibody levels against aMPV were observed in birds vaccinated with virosomes. These results demonstrate that immunization of turkeys with aMPV virosomes can be an effective strategy for control of disease.     

Emergence of a virulent type C avian metapneumovirus in turkeys in Minnesota

The objectives of the present study were to investigate the pathogenesis of a recent isolate of avian metapneumovirus (aMPV) in turkeys and to evaluate the quantitative distribution of the virus in various tissues during the course of infection. Seventy 2-week-old turkey poults were divided equally into two groups. One group was inoculated with aMPV (MN 19) with a titer of 10(5.5) TCID50 oculonasally. Birds in the second group were maintained as sham-inoculated controls. Birds showed severe clinical signs in the form of copious nasal discharge, swollen sinus, conjunctivitis, and depression from 4 days postinoculation (PI) to 12 days PI. Samples from nasal turbinates, trachea, conjunctiva, Harderian gland, infraorbital sinus, lungs, liver, and spleen were collected at 1, 3, 5, 7, 9, 11, and 14 days PI. Histopathologic lesions such as a multifocal loss of cilia were prominent in nasal turbinate and were seen from 3 to 11 days PI. Immunohistochemistry revealed the presence of aMPV from 3 to 9 days PI in nasal turbinate and trachea. Viral RNA could be detected for 14 days PI from nasal turbinate and for 9 days from trachea. In situ hybridization demonstrated the presence of aMPV from 1 to 11 days PI in nasal turbinates and from 3 to 9 days PI in the trachea. Quantitative real-time polymerase chain reaction data showed the presence of a maximum amount of virus at 3 days PI in nasal turbinate and trachea. Clinically and histopathologically, the new isolate appears to be more virulent compared to the early isolates of aMPV in the United States.     

Alloimmunity does not protect from challenge with the feline immunodeficiency virus

Immune responses against polymorphic host molecules incorporated into lentiviral envelopes during cell budding have induced protection against primate immunodeficiency virus infection. Dendritic cells (DCs) express high levels of MHC molecules and are infectable by lentiviruses. Therefore, in this pilot study we addressed the hypothesis that immunization of cats with allogeneic DC would induce immune responses that protect against challenge with the feline immunodeficiency virus. Two groups of 3 cats each received 3 subcutaneous injections of allogeneic or autologous DC, and were then challenged with viruses propagated in the immunizing DC. Infection status and lymphocyte parameters of cats were assessed during 6 weeks after challenge. MHC II antigens were incorporated into viral particles as identified by Western blot; and antibodies reactive with MHC class II antigens were detected in the serum of cats immunized with allogeneic but not autologous DC. After challenge, all cats had proviral DNA in blood leukocytes from 2 weeks post-challenge onward and seroconverted. Cats immunized with allogeneic DC maintained higher total and CD21(+) lymphocyte concentrations, and higher CD4(+)/CD8(+) lymphocyte ratios; however, these differences were not significantly different from cats that received autologous DC immunizations. Plasma viral load was not significantly different between groups of cats (p=0.204). These results suggest that immunization of cats with allogeneic DC does not induce protective immunity against FIV infection.     

Isolation and characterization of a subtype C avian metapneumovirus circulating in Muscovy ducks in China

Subtype C avian metapneumovirus (aMPV-C), is an important pathogen that can cause egg-drop and acute respiratory diseases in poultry. To date, aMPV-C infection has not been documented in Muscovy ducks in China. Here, we isolated and characterized an aMPV-C, designated S-01, which has caused severe respiratory disease and noticeable egg drop in Muscovy duck flocks in south China since 2010. Electron microscopy showed that the isolate was an enveloped virus exhibiting multiple morphologies with a diameter of 20–500 nm. The S-01 strain was able to produce a typical cytopathic effect (CPE) on Vero cells and cause death in 10- to 11-day-old Muscovy duck embryos. In vivo infection of layer Muscovy ducks with the isolate resulted in typical clinical signs and pathological lesions similar to those seen in the original infected cases. We report the first complete genomic sequence of aMPV-C from Muscovy ducks. A phylogenetic analysis strongly suggested that the S-01 virus belongs to the aMPV-C family, sharing 92.3%-94.3% of nucleotide identity with that of aMPV-C, and was most closely related to the aMPV-C strains isolated from Muscovy ducks in France. The deduced eight main proteins (N, P, M, F, M2, SH, G and L) of the novel isolate shared higher identity with hMPV than with other aMPV (subtypes A, B and D). S-01 could bind a monoclonal antibody against the F protein of hMPV. Together, our results indicate that subtype-C aMPV has been circulating in Muscovy duck flocks in South China, and it is urgent for companies to develop new vaccines to control the spread of the virus in China.     

Production and characterization of monoclonal antibodies that react to the nucleocapsid protein of avian metapneumovirus subtype C

Monoclonal antibodies (MAbs) were prepared against avian metapneumovirus (aMPV) subtype C (aMPV/ Minnesota/turkey/1a/97). Six MAbs were selected based on enzyme-linked immunosorbent assay activities and characterized by isotyping, neutralization test, western blot analysis, and immunohistochemistry assay. The results showed that three MAbs (3E, 9D, and 12C) belonged to the IgG1 subclass, whereas the other three (5D, 8E, and 16E) were of the IgG2a subclass. None of the six MAbs neutralized aMPV infectivity at a detectable level, but all reacted with both denatured and nondenatured forms of the nucleocapsid (N) protein of aMPV, suggesting that these MAbs may recognize structurally independent epitopes of the N protein. These MAbs provide new tools and methods for investigating aMPV infection and pathogenesis, as well as diagnosis of aMPV disease.     

Protection by recombinant viral proteins against a respiratory challenge with virulent avian metapneumovirus

Protection by recombinant avian metapneumovirus (aMPV) N or M proteins against a respiratory challenge with virulent aMPV was examined. N, M or N+M proteins were administered intramuscularly (IM) with incomplete Freund's adjuvant (IFA) or by the oculonasal (ON) route with cholera toxin-B (CTB). Each turkey received 40 or 80 microg of each recombinant protein. Birds were considered protected against challenge if the challenge virus was not detectable in the choanal swabs by RT-PCR. At a dose of 40 microg/bird, N protein given with IFA by the IM route protected eight out of nine birds. M protein at the same dose protected three out of seven birds, while a combination of N+M proteins (40 microg each) protected three out of four birds. At a dose of 80 microg of each of N and M proteins per bird given with IFA by the IM route, 100% protection was achieved. ON immunization with a mixture of N and M proteins induced partial protection when the proteins were given with CTB; no detectable protection was noted without CTB. N and M proteins induced anti-aMPV antibodies, although protection against virulent virus challenge did not appear to be associated with the level or presence of antibodies.     

Immune response of poults following intranasal inoculation with Artvax vaccine and a formalin-inactivated Bordetella avium bacterin

Poults 3 weeks and older developed temporary tracheal resistance to intranasal challenge following inoculation of either Artvax vaccine or formalin-inactivated Bordetella avium bacterin by the intranasal and eyedrop routes. Resistance usually persisted for 3-4 weeks after B. avium challenge. However, with constant exposure to infected controls, the vaccinated birds eventually developed tracheal infection. Day-old poults did not respond to either the Artvax or the bacterin and were completely susceptible to challenge. Two-week-old poults responded to some degree, but poults 3 weeks old and older responded best. Poults inoculated with bacterin by aerosol or by drinking water did not respond as well as those inoculated by the intranasal and eyedrop routes. When poults were given a single subcutaneous injection at 3 weeks of age and challenged 2 weeks later, three of five resisted infection for 18 days.     

Mucosal immunization of piglets with purified F18 fimbriae does not protect against F18+ Escherichia coli infection

Post-weaning diarrhoea and oedema disease in weaned piglets are caused by infection with F4⁺ or F18⁺ Escherichia coli strains. There is no commercial vaccine available, but it is shown that oral immunization of weaned piglets with purified F4 fimbriae induces a protective mucosal immune response. In the present study, piglets were orally and nasally immunized with purified F18 fimbriae in the presence of the mucosal adjuvant LT(R192G) or CTA1-DD, respectively. This immunization could not lead to protection against F18⁺ E. coli infection. The induced F18-specific immune response was directed towards the major subunit FedA and weakly towards the adhesive subunit FedF. The results of these experiments demonstrate that it is difficult to induce protective immunity against F18+ E. coli using the whole fimbriae due to the low response against the adhesin.     

Immunization of turkeys with a DNA vaccine expressing either the F or N gene of avian metapneumovirus

In this study we compared protection by DNA vaccination with the F (pCMV-F) or N (pCMV-N) gene from avian metapneumovirus (aMPV) in turkeys. One-week-old turkey poults received two intramuscular injections 2 wk apart. Birds were challenged with a turkey-embryo-adapted aMPV at 5 wk of age. Birds vaccinated with pCMV-F had decreased clinical signs of disease as well as significantly reduced virus load in tracheal swabs compared with birds vaccinated with pCMV-N or unvaccinated control birds. Serum neutralizing antibodies were significantly higher in birds receiving pCMV-F compared with all other groups. These results indicate that DNA vaccination with the F, but not N, gene of aMPV can induce significant protection against aMPV infection.     

La Sota vaccination may not protect against virus shedding and the lesions of velogenic Newcastle disease in commercial turkeys

The aim of this project is to study the clinical signs and lesion of velogenic Newcastle disease (vND) in commercial turkeys, and also to find out if La Sota vaccination offered protection against these signs and lesions. The cockerels were included as positive controls. One hundred and twenty turkey poults and cockerels were divided into eight groups as follows: unvaccinated unchallenged turkeys (UUT), unvaccinated challenged turkeys (UCT), vaccinated unchallenged turkeys (VUT), vaccinated challenged turkeys (VCT), and along the same lines, the cockerel groups were UUC, UCC, VUC and vaccinated challenged cockerels (VCC). Vaccination was at 3 weeks of age while challenge was at 6 weeks of age. The unvaccinated turkeys and cockerels (UCT and UCC) showed different degrees of depression, diarrhoea and later paralysis at challenge. Total mortality was 100% in cockerels within 6 days, but 60% in turkeys. Similar but milder clinical signs were found in the VCC with a total mortality of 13.3%. The VCT showed mild drop in feed and water consumption, and no mortality. All the challenged groups had significant (p < 0.05) loss of weight when compared with their controls. Necropsy showed that while the UCC had severe proventricular haemorrhages, intestinal and caecal tonsil ulcers, the UCT had no digestive tract lesion. There was severe atrophy of the lymphoid organs in all the challenged groups. Histopathological sections of the bursa, spleen and thymus in all the challenged groups with special emphasis on the vaccinated and unvaccinated turkeys with mortalities of 0 and 60%, respectively, had very severe necrosis and depletion of the lymphoid tissue. Virus was isolated from the cloacal swabs. The haemagglutination inhibition antibodies were significantly higher (p < 0.05) in the challenged groups than the unchallenged. The above observations in the intestinal tracts of UCT are of diagnostic significance while the gross and microscopic lesions in the UCT and VCT show that La Sota vaccination may not protect turkeys against the destruction of the lymphoid organs by vND as earlier reported in chickens. This may lead to immunosuppression and production problems in areas where vND is enzootic.     

Use of ovotransferrin as an antimicrobial in turkeys naturally infected with Chlamydia psittaci, avian metapneumovirus and Ornithobacterium rhinotracheale

Respiratory pathogens are difficult to control in large-scale turkey production. This report describes a clinical trial of antimicrobial ovoTF aerosol on a large Belgian turkey farm. ovoTF was administered to reduce Chlamydia psittaci (C. psittaci) infections and to study the impact of this action on the occurrence of Ornithobacterium rhinotracheale (O. rhinotracheale) and avian metapneumovirus (aMPV) infections. Two subsequent broods were included; (i) a control brood receiving no ovoTF and (ii) an ovoTF brood receiving ovoTF aerosol (5mg/animal) at the age of 2 weeks, continuing daily for 12 days. Twenty-four one-day-old toms of the control and ovoTF brood were tagged and monitored for 15 weeks. The control brood experienced two periods of respiratory disease, the first (2-3 weeks of age) due to C. psittaci and the second (8-17 weeks of age) in the presence of C. psittaci, O. rhinotracheale and maybe aMPV. Extensive antibiotic treatment was needed in 2, 8 and 9 week-old toms. In the ovoTF brood, toms stayed healthy until the age of 9 weeks, whereafter respiratory disease occurred in the presence of C. psittaci, O rhinotracheale and aMPV. OvoTF administration: (i) reduced the amount of C. psittaci in the air as demonstrated by bioaerosol monitoring, (ii) prevented respiratory disease during the first half of the brood period, (iii) was associated with 46% reduction of mortality, and (iv) reduced the antibiotic cost. Our results justify additional clinical trials to explore the use of this innovative antimicrobial strategy for poultry.     

Clinical and immunological responses of ewes following vaccination with an experimental formalin-inactivated Chlamydia psittaci (ovis) vaccine and subsequent challenge with the live organism during pregnancy

The protection afforded by an experimental, killed, adjuvanted vaccine derived from the A22 strain of Chlamydia psittaci (ovis) against ovine enzootic abortion was studied. The vaccine was used undiluted (group A), at a dilution of 10(-3) (group B) and at a dilution of 10(-6) (group C). A fourth control group (group D) was inoculated with all components of the vaccine except the chlamydial antigen. A group of rams (group R) was also vaccinated with the chlamydial antigen diluted to 10(-3). Animals were challenged 70 days after mating with the A22 strain of C. psittaci (ovis) and were studied throughout pregnancy and the subsequent lambing period. Their cell-mediated immune responses were examined using a skin test and their humoral immune responses were studied using an ELISA. Tests for excretion of chlamydiae in their faeces and genital tract during pregnancy and after parturition and in the faeces of their lambs were made. The reproductive performance of the ewes was assessed by calculating the average weight of lambs produced per ewe in each group. The experimental vaccine protected the ewes in groups A and B against challenge with C. psittaci (ovis) as none showed clinical signs of OEA or excreted chlamydiae. The average weight of lambs produced per ewe in both groups was greater than 4 kg. Both groups seroconverted after vaccination but not all of them were positive to the skin test. The experimental vaccine at 10(-6) dilution of antigen did not protect the ewes as three of 10 ewes displayed clinical OEA and excreted chlamydiae in the products of parturition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Passive immunization with convalescent serum, or oral immunization with formalin-killed organisms, does not protect mice against gastrointestinal challenge with Listeria monocytogenes

Listeria monocytogenes is an important foodborne pathogen in both humans and animals. In addition, murine listeriosis is a widely used model for studying the molecular pathogenesis of an intracellular pathogen, and the regulation of protective cellular immunity. Little attention has been paid to protective immunity against L. monocytogenes in the gastrointestinal tract, where a secretory immune response might prevent attachment of the bacteria to the intestinal epithelium. In this study we found that neither opsonization of L. monocytogenes with immune serum, nor repeated oral administration of killed L. monocytogenes, protected mice against gastrointestinal challenge with L. monocytogenes.     

A single vaccination of commercial broilers does not reduce transmission of H5N1 highly pathogenic avian influenza

ABSTRACT: Vaccination of chickens has become routine practice in Asian countries in which H5N1 highly pathogenic avian influenza (HPAI) is endemically present. This mainly applies to layer and breeder flocks, but broilers are usually left unvaccinated. Here we investigate whether vaccination is able to reduce HPAI H5N1 virus transmission among broiler chickens. Four sets of experiments were carried out, each consisting of 22 replicate trials containing a pair of birds. Experiments 1-3 were carried out with four-week-old birds that were unvaccinated, and vaccinated at day 1 or at day 10 of age. Experiment 4 was carried out with unvaccinated day-old broiler chicks. One chicken in each trial was inoculated with H5N1 HPAI virus. One chicken in each trial was inoculated with virus. The course of the infection chain was monitored by serological analysis, and by virus isolation performed on tracheal and cloacal swabs. The analyses were based on a stochastic SEIR model using a Bayesian inferential framework. When inoculation was carried out at the 28th day of life, transmission was efficient in unvaccinated birds, and in birds vaccinated at first or tenth day of life. In these experiments estimates of the latent period (~1.0 day), infectious period (~3.3 days), and transmission rate parameter (~1.4 per day) were similar, as were estimates of the reproduction number (~4) and generation interval (~1.4 day). Transmission was significantly less efficient in unvaccinated chickens when inoculation was carried out on the first day of life. These results show that vaccination of broiler chickens does not reduce transmission, and suggest that this may be due to the interference of maternal immunity.