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We previously reported that recombinant Newcastle disease virus LaSota (rLS) expressing infectious bronchitis virus (IBV) Arkansas (Ark)-type trimeric spike (S) ectodomain (Se; rLS/ArkSe) provides suboptimal protection against IBV challenge. We have now developed rLS expressing chicken granulocyte-macrophage colony-stimulating factor (GMCSF) and IBV Ark Se in an attempt to enhance vaccine effectiveness. In the current study, we first compared protection conferred by vaccination with rLS/ArkSe and rLS/ArkSe.GMCSF. Vaccinated chickens were challenged with virulent Ark, and protection was determined by clinical signs, viral load, and tracheal histomorphometry. Results showed that coexpression of GMCSF and the Se from rLS significantly reduced tracheal viral load and tracheal lesions compared with chickens vaccinated with rLS/ArkSe. In a second experiment, we evaluated enhancement of cross-protection of a Massachusetts (Mass) attenuated vaccine by priming or boosting with rLS/ArkSe.GMCSF. Vaccinated chickens were challenged with Ark, and protection was evaluated. Results show that priming or boosting with the recombinant virus significantly increased cross-protection conferred by Mass against Ark virulent challenge. Greater reductions of viral loads in both trachea and lachrymal fluids were observed in chickens primed with rLS/ArkSe.GMCSF and boosted with Mass. Consistently, Ark Se antibody levels measured with recombinant Ark Se protein-coated ELISA plates 14 days after boost were significantly higher in these chickens. Unexpectedly, the inverse vaccination scheme, that is, priming with Mass and boosting with the recombinant vaccine, proved somewhat less effective. We concluded that a prime and boost strategy by using rLS/ArkSe.GMCSF and the worldwide ubiquitous Mass attenuated vaccine provides enhanced cross-protection. Thus, rLS/GMCSF coexpressing the Se of regionally relevant IBV serotypes could be used in combination with live Mass to protect against regionally circulating IBV variant strains.Protección incrementada por el virus recombinante de la enfermedad de Newcastle que expresa el ectodominio de la espícula del virus de la bronquitis infecciosa y el factor estimulante de colonias de granulocitos y macrófagos del pollo. Anteriormente se reportó que la cepa LaSota recombinante del virus de la enfermedad de Newcastle (rLS) que expresa el ectodominio de la espícula trimérica (S) de tipo Arkansas (Ark) del virus de la bronquitis infecciosa (IBV) (Se; rLS/ArkSe) proporciona una protección subóptima contra la exposición al virus de la bronquitis infecciosa. Ahora se ha desarrollado hemos desarrollado una cepa LaSota recombinante (rLS) que expresa el factor estimulante de colonias de granulocitos y macrófagos de pollo (GMCSF) y la espícula del virus de bronquitis Arkansas en un intento para mejorar la efectividad de la vacuna. En el estudio actual, primero se comparó la protección conferida por la vacunación con los virus rLS/ArkSe y rLS/ArkSe.GMCSF. Los pollos vacunados se desafiaron con un virus Arkansas virulento y la protección se determinó mediante los signos clínicos, la carga viral y la histomorfometría de la tráquea. Los resultados mostraron que la coexpresión del factor estimulante de colonias de granulocitos y macrófagos de pollo y la espícula de la cepa recombinante LaSota redujo significativamente la carga viral traqueal y las lesiones traqueales en comparación con los pollos vacunados con el virus rLS/ArkSe. En un segundo experimento, se evaluó el incremento en la protección cruzada por una vacuna atenuada de Massachusetts (Mass) mediante la primovacunación o la vacunación de refuerzo con rLS/ArkSe.GMCSF. Los pollos vacunados fueron desafiados con el virus Arkansas y se evaluó la protección. Los resultados mostraron que la primovacunación o la vacunación de refuerzo con el virus recombinante aumentó significativamente la protección cruzada conferida por el virus Massachusetts contra el desafío virulento con el virus Arkansas. Se observaron mayores reducciones de las cargas virales en los fluidos traqueales y lagrimales en pollos primovacunadoss con rLS/ArkSe.GMCSF y con vacunación de refuerzo con Massachusetts. De manera consistente, los niveles de anticuerpos Ark Se medidos con placas de ELISA recubiertas con proteína Ark Se recombinante a los 14 días después del refuerzo fueron significativamente más altos en estos pollos. De manera inesperada, el esquema de vacunación inverso, es decir, la primovacunación con Massachusetts y el refuerzo con la vacuna recombinante, resultó menos efectivo. Se concluye que una estrategia de primovacunación y refuerzo mediante el uso de rLS/ArkSe.GMCSF y la vacuna atenuada con Massachusetts usada en todo el mundo proporciona una protección cruzada aumentada. Por tanto, el virus rLS/GMCSF que coexpresa la proteína de la espícula de los serotipos regionales relevantes de bronquitis infecciosa podría usarse en combinación con una vacuna viva Massachusetts para proteger contra cepas variantes del virus de la bronquitis infecciosa que circulan regionalmente.

Severity of the tracheal histologic inflammatory response induced in broilers by ocular inoculation of two infectious bronchitis (IBV) and three Newcastle disease virus (NDV) commercial vaccines were evaluated. The vaccine was delivered by eye drop with a coarse spray to day-old chicks. The vaccines were given individually or in various combinations and were evaluated relative to nonvaccinated controls. Evaluations were performed on postvaccination (PV) days 7 and 14. Histologic endpoints included semiquantitative severity scoring of inflammatory components and quantitative morphometric determinations of inflammatory cell concentration, mucosal thickness, and percentage of ciliated mucosal surface. Strong positive correlations were observed between routine severity scoring and morphometric inflammatory parameters, whereas a negative correlation was present between inflammation severity and the percentage of mucosal ciliation. Variable, sometimes extensive, and often statistically significant differences in inflammatory responses were observed between the various vaccines. One IBV Massachusetts strain vaccine (IBV-A) produced the greatest overall inflammatory response when given alone or in combination with the NDV vaccines. Enhancement of tracheitis was seen on PV day 14 by covaccination of IBV-A with the NDV vaccines, but not by covaccination of another IBV Massachusetts strain vaccine (IBV-B) with NDV. Reduction in cilia percentage was observed for all vaccine groups relative to controls on PV day 7. However, although reactive cilia regeneration occurred on PV day 14 for most vaccine groups, a cilia regenerative response was not apparent for individual or NDV combination vaccination for IBV-A. The study also demonstrates that substantial microscopic trachea pathology may be present in vaccinated birds not exhibiting apparent clinical respiratory signs.

Avian infectious bronchitis viruses (IBVs) with the TC07-2 genotype have spread rapidly in East Asia since they were first reported in China in 2007. In 2015, an IBV with the TC07-2 genotype (designated KrD1515) was isolated from layer chickens with severe respiratory symptoms in Korea. In the present study, the full-length open reading frames of the spike (S) and nucleocapsid (N) genes of the virus were sequenced and analyzed. S1 gene phylogenetic analysis revealed that the KrD1515 virus clustered with viruses with the TC07-2 genotype, whereas N gene phylogenetic analysis revealed that the KrD1515 virus clustered with Korean IBVs, but not with Chinese TC07-2 IBV. When 7-day-old specific-pathogen-free chickens were inoculated with the KrD1515 virus, they developed severe respiratory symptoms and tracheal lesions. However, there were no other clinical symptoms or pathologic lesions in other tissues. The virus was shed from the trachea for at least a week and from the cloaca for only a day. Our findings suggest that the KrD1515 virus is a recombinant virus between a Chinese TC07-2 IBV and a non-TC07-2 Korean IBV and engages in respiratory tropism in chickens.

Infectious laryngotracheitis (ILT) is a highly contagious disease of chickens and is responsible for significant economic losses in the poultry industry worldwide; it is caused by Gallid herpesvirus-1 (GaHV-1), commonly known as infectious laryngotracheitis virus (ILTV). Experimental evaluation of ILTV strains is fundamental to identify changes in virulence that can contribute to the severity and spread of outbreaks and consequently influence the efficacy of vaccination. Several criteria had been utilized to determine the degree of virulence associated with ILTV strains. The objectives of this study were to compare the levels of virulence of the standard United States Department of Agriculture (USDA) challenge strain with a contemporary outbreak-related strain (63140) and to evaluate the efficacy of individual criteria to identify changes in virulence. Broilers were inoculated with increasing infectious doses of each strain. The criteria utilized to evaluate virulence were clinical signs of the disease, mortality, microscopic tracheal lesions, trachea genome viral loads, and antibody titers. Clinical signs scores were a useful parameter to define the peak of clinical disease but did not reveal differences in virulence between strains. Similarly, trachea microscopic lesion scores or levels of serum antibody titers were parameters that did not reveal obvious differences in virulence between strains. However, mortalities and increased viral genome loads in trachea of chickens inoculated with lower (log10 1 to 2) infectious doses clearly differentiated 63140 as a more-virulent ILTV strain. This study provides the framework to compare the virulence level of emerging ILTV isolates to the now-characterized USDA and 63140 strains.

Factors responsible for the persistence of Arkansas Delmarva Poultry Industry (ArkDPI)-derived infectious bronchitis vaccines in commercial flocks and the high frequency of isolation of ArkDPI-type infectious bronchitis viruses in respiratory cases are still unclear. We compared dynamics of vaccine viral subpopulations, viral loads, persistence in trachea and cloaca, and the magnitude of infectious bronchitis virus (1BV)-specific antibody induction after vaccination with two commercial ArkDPI-derived Arkansas (Ark) serotype vaccines. One of the vaccines (coded vaccine B) produced significantly higher vaccine virus heterogeneity in vaccinated chickens than the other vaccine (coded A). Chickens vaccinated with vaccine B had significantly higher viral loads in tears at 5 days postvaccination (DPV) than those vaccinated with vaccine A. Vaccine B also induced a significantly higher lachrymal immunoglobulin M response at 11 DPV, an earlier peak of IBV-specific lachrymal immunoglobulin A, and higher serum antibodies than vaccine A. In addition, a significantly higher proportion of birds vaccinated with vaccine B had vaccine virus detected in the trachea at 20 DPV than those vaccinated with vaccine A. Furthermore, the virus detected at 20 DPV in most of the chickens vaccinated with vaccine B was a single specific subpopulation (subpopulation 4) selected from multiple vaccine subpopulations detected earlier at 5 and 7 DPV in the same chickens. On the other hand, a higher proportion of chickens vaccinated with vaccine A had virus detected in cloacal swabs at 20 DPV. Thus we found differences in mucosal antibody induction and selection and persistence of vaccine viruses between two ArkDPI-derived vaccines from different manufacturers. The higher vaccine virus heterogeneity observed in chickens vaccinated with vaccine B compared with those vaccinated with vaccine A may be responsible for these differences. Thus the high frequency of Ark IBV viruses in the field may be due to the inherent ability of some ArkDPI-derived vaccine viruses to be selected and persist in vaccinated chickens. Vaccine virus persistence may offer genetic material for recombination or may undergo mutations with the potential to result in increased virulence.

Infectious bronchitis virus (IBV) infections cause great economic losses to the poultry industry worldwide. IBVs continuously evolve by developing mutations in antigenic sites; therefore, an IBV vaccine that provides broad cross-protection can be a highly relevant and practical method in IBV control strategies. Although some IBV vaccine strains are known to provide protection against multiple IBV serotypes, in general commercially available IBV vaccine strains provide protection against antigenically related viruses but not distinct heterologous viruses. In the present study we characterized the Korean variant IBV K40/09 strain with regard to its immunogenicity and protective efficacy against seven currently circulating IBV serotypes. Three-week-old specific-pathogen-free chickens were intraocularly immunized with the IBV K40/09 strain at 10(3.5) 50% egg infective dose (EID50). Three weeks after immunization all the birds were challenged with seven different strains at 10(4.5) EID50. Chickens immunized with the IBV K40/09 strain showed significantly high levels of protection against all challenge viruses at the trachea and kidney levels. Our results suggest that IBV K40/09 could be useful to ensure IBV vaccine effectiveness owing to its cross-protective ability. Therefore, the IBV K40/09 strain merits consideration as a vaccine candidate to prevent infection as well as the spread of new IBV strains and many IBV variants that have been reported worldwide.

Infectious bronchitis coronavirus (IBV) shows extensive genotypic and phenotypic variability. The evolutionary process involves generation of genetic diversity by mutations and recombination followed by replication of those phenotypes favored by selection. In the current study, we examined changes occurring in a wild Arkansas (Ark) challenge strain in chickens that were vaccinated either ocularly with commercially available attenuated ArkDPI-derived vaccines or in ovo with a replication-defective recombinant adenovirus expressing a codon-optimized IBV Ark S1 gene (AdArkIBV.S1(ck)). Commercial IBV Ark vaccines A, B, and C provided slightly differing levels of protection against homologous challenge. Most importantly for the current study, chickens vaccinated with the different vaccines displayed significant differences in specific B-lymphocyte responses in the Harderian gland (i.e., the challenge virus encountered differing immune selective pressure during invasion among host groups). Based on S1 sequences, five predominant populations were found in different individual vaccinated/challenged chickens. Chickens with the strongest immune response (vaccine A) were able to successfully impede replication of the challenge virus in most chickens, and only the population predominant in the challenge strain was detected in a few IBV-positive birds. In contrast, in chickens showing less than optimal specific immune responses (vaccines B and C) IBV was detected in most chickens, and populations different from the predominant one in the challenge strain were selected and became predominant. These results provide scientific evidence for the assumption that poor vaccination contributes to the emergence of new IBV strains via mutation and/or selection. In ovo vaccination with a low dose of AdArkIBV.S1(ck) resulted in a mild increase of systemic antibody and reduced viral shedding but no protection against IBV signs and lesions. Under these conditions we detected only virus populations identical to the challenge virus. Possible explanations are discussed. From a broad perspective, these results indicate that selection is an important force driving IBV evolution.

Recent reports have shown an increased recovery of cells from flocked nylon swabs which may improve the specimen quality and the real sensitivity of diagnostic tests in a clinical setting. In this study, the detection of Mycoplasma gallisepticum (MG) and M. synoviae (MS), using dry swabs of different materials (nylon flocked, cotton, and polyester), was investigated using real-time TaqMan PCR protocols. Different types of samples, including dilutions of pure broth cultures of MG and MS as well as swabs from tracheas of experimentally infected chickens and field cases of infection, were analyzed. There were no statistical differences in real-time PCR results among the different swab types (P < 0.05), indicating that this is not likely to be a significant factor in MG and MS detection by this method.

Highly pathogenic (HP) H5N1 avian influenza (AI) is enzootic in several countries of Asia and Africa and constitutes a major threat, at the world level, for both animal and public health. Ducks play an important role in the epidemiology of AI, including HP H5N1 AI. Although vaccination can be a useful tool to control AI, duck vaccination has not proved very efficient in the field, indicating a need to develop new vaccines and a challenge model to evaluate the protection for duck species. Although Muscovy duck is the duck species most often reared in France, the primary duck-producing country in Europe, and is also produced in Asia, it is rarely studied. Our team recently demonstrated a good cross-reactivity with hemagglutinin from clade 2.2 and inferred that this could be a good vaccine candidate for ducks. Two challenges using two French H5N1 HP strains, 1) A/mute swan/France/06299/06 (Swan/06299), clade 2.2.1, and 2) A/mute swan/France/070203/07 (Swan/070203), clade 2.2 (but different from subclade 2.2.1), were performed (each) on 20 Muscovy ducks (including five contacts) inoculated by oculo-nasal route (6 log10 median egg infectious doses per duck). Clinical signs were recorded daily, and cloacal and oropharyngeal swabs were collected throughout the assay. Autopsies were done on all dead ducks, and organs were taken for analyses. Virus was measured by quantitative reverse transcriptase-PCR based on the M gene AI virus. Ducks presented severe nervous signs in both challenges. Swan/070203 strain led to 80% morbidity (12/15 sick ducks) and 73% mortality (11/15 ducks) at 13.5 days postinfection (dpi), whereas Swan/06299 strain produced 100% mortality at 6.5 dpi. Viral RNA load was significantly lower via the cloacal route than via the oropharyngeal route in both trials, presenting a peak in the first challenge at 3.5 dpi and being more stable in the second challenge. The brain was the organ containing the highest viral RNA load in both challenges. Viral RNA load in a given organ was similar or statistically significantly higher in ducks challenged with Swan/06299 strain. Thus, the Swan/06299 strain was more virulent and could be used as a putative challenge model. Moreover, challenged ducks and contacts contained the same amounts of viral RNA load, demonstrating the rapid and efficient transmission of H5N1 HP in Muscovy ducks in our experimental conditions.

Increased mortality was reported in two flocks of Muscovy ducklings from two consecutive hatches originating from the same breeder flock. Coughing, dyspnea, and gasping were observed in some ducklings between 6 and 11 days of age. Opaque white plugs of exudate were seen in the tracheas with some ducklings having multiple tracheal plugs. Tracheal and bronchial epithelium was hyperplastic and superficial epithelial cells contained eosinophilic intranuclear viral inclusions. Virus particles compatible with adenovirus morphology were observed in tracheal epithelial cells by electron microscopy and in the supernatant from cell cultures inoculated with filtered tracheal homogenates. The isolated virus was genetically indistinguishable from duck adenovirus 1 (DAdV-1). Our report confirms for the first time the presence of DAdV-1 in Canada and also reports for the first time adenovirus-associated respiratory disease in ducklings and supports previous findings that some DAdV-1 can be pathogenic even in waterfowl.

We compared detection of infectious bronchitis virus (IBV) by quantitative RT-PCR (qRT-PCR) in tears and trachea of IBV-infected chickens and found that quantitative detection of IBV RNA in tears is more sensitive than in tracheal homogenates. Furthermore, we demonstrated that IBV contained in chicken lachrymal fluid is infectious and that tears of IBV-infected chickens can be used to infect naive chickens. We compared the immune responses to IBV in the Harderian gland and cecal tonsils of immunocompetent chickens and chickens infected with chicken anemia virus (CAV) and/or infectious bursal disease virus (IBDV). Flow cytometry analyses of lymphocytes in Harderian glands and cecal tonsils indicated that the relative abundance of IgM+ B cells in the Harderian glands and cecal tonsils following exposure to IBV in combination with immunosuppressive viruses was reduced compared to chickens infected with IBV alone. CAV, but not IBDV, reduced the CD4+/CD8+ T cell ratios compared to chickens infected with IBV alone. Enzyme-linked immuno-spot forming assays on cells in the Harderian glands and cecal tonsils of IBV-infected chickens indicated that maximum IBV-specific IgA-secreting cell responses were reduced in chickens infected with CAV. IBDV co-infected chickens displayed a delayed IgA response to IBV. Thus immunosuppressive viruses reduced B cells and T helper cells in the Harderian glands and cecal tonsils in response to IBV, and slowed the kinetics and/or reduced the magnitude of the mucosal immune response against IBV. We have shown for the first time that CAV affects pathogen-specific B cell responses in a mucosal effector site.

Various diagnostics techniques were compared for their ability to detect infectious laryngotracheitis (ILT) during an outbreak in chickens aged between 4 and 21 wk. Gross lesions ranged from excess mucus to accumulation of fibrinonecrotic exudate in the larynx and trachea. Syncytial cells with intranuclear inclusion bodies were found in sinus, conjunctiva, larynx, trachea, lung, and air sac. Virus isolation in chicken embryos was attempted in every case. Negative-stain electron microscopy detected herpesvirus in only 6% of the cases. Yet, isolation of ILT virus in the chorioallantoic membrane was presumed by histology in20% of the samples and confirmed by fluorescent antibody (FA) in 35% of the embryos inoculated with conjunctivas or tracheas from affected birds. Overall, results from histology and FA tests were highly correlated. FA test has the advantage over histology of being diagnostically specific for ILT virus. Polymerase chain reaction was the most sensitive test and detected the viral DNA even in cases where histology and FA were negative. ILT virus DNA was quantified by real-time polymerase chain reaction (Re-Ti ILTV). Histologic and FA results from larynx and trachea were negative if the concentration of the viral DNA wasor =4 of log10. A viral DNA concentration higher than log10 4, as determined by Re-Ti ILTV, was required for clinical ILT to be manifested.

In this study, we examined the association among clinical signs, ciliostasis, virus detection, and histopathology for evaluating protection of vaccinated chickens against homologous and heterologous infectious bronchitis virus (IBV) challenge. At 5 days following challenge with IBV, we found a good correlation among clinical signs, ciliostasis in the trachea, challenge virus detection, and microscopic lesions in the trachea, with all four criteria being negative in fully protected birds and positive in fully susceptible birds. In partially protected birds we observed clinical signs and detected challenge virus; however, the ciliated epithelium was intact. In a second experiment, we challenged fully protected, partially protected, and fully susceptible birds with IBV, and then at 5 days postchallenge we gave the birds an opportunistic bacterium intranasally. Twenty Bordetella avium colonies were recovered from one of five fully protected birds, and only five colonies were isolated from two of five partially protected birds without ciliostasis, whereas in birds with ciliostasis, numerous colonies were isolated. Obviously, decreasing IBV infection and replication in the upper respiratory tract will decrease transmission and mutations, leading to variant viruses, and herein we demonstrate that protection of the cilia will decrease secondary bacterial infections, which have been shown to lead to condemnations and increased mortality. Thus, it appears that examining both criteria would be important when evaluating IBV vaccine efficacy.

The onset of protective immunity with MS-H was determined through experimental challenge and compared with the parent strain 86079/7NS. MS-H vaccinates and 86079/7NS inoculates were challenged at 1, 2, 3, 4, 5, and 6 wk after vaccination, then examined 2 wk after challenge for signs of respiratory disease. Serologic results indicated that 100% of MS-H vaccinates had antibodies to MS by 3 wk after vaccination and 100% of 86079/7NS inoculates were positive by 2 wk after inoculation. From 3 wk after vaccination, MS-H vaccinates had a significantly lower incidence of air sac lesions and, from 4 wk after vaccination, a significantly lower air sac lesion severity. In 86079/7NS-inoculated birds, a significantly lower incidence of air sac lesions was observed from 1 wk after inoculation, and air sac lesion severity was significantly lower than the unvaccinated controls at 3 wk after inoculation. It would appear that, under the conditions of this experiment, protective immunity elicited by MS-H appeared at 4 wk after vaccination, slightly later than the appearance of serum antibody. Although the MS-H vaccine was slower to establish protective immunity than 86079/7NS, there was no significant difference between the two strains by 4 wk after vaccination or inoculation.

The objective of this study was to compare the presence of the Arkansas (Ark) and Massachusetts (Mass) serotypes of infectious bronchitis virus (IBV) in the tracheas and cecal tonsils of commercial broilers after vaccination at 1 day of age by coarse spray. When given as a single serotype vaccine, the Mass strain was detected by reverse transcriptase-polymerase chain reaction (RT-PCR)-restriction fragment length polymorphism (RFLP) only in the tracheas, whereas the Ark strain was detected in both the tracheas and cecal tonsils. By in situ hybridization, the Mass and Ark nucleocapsid (Nc) genes were detected only at 7 days in the tracheas. When both strains were given in the mixed vaccine, the Mass strain was more consistently detected by RT-PCR-RFLP in the tracheas and cecal tonsils at early stages of infection (up to 14 days) and the Arkansas strain was more consistently detected at late stages of infection (21 and 28 days). By in situ hybridization, the IBV Nc gene was more consistently detected in the trachea at early stages of infection (7, 14, and 21 days) and in the cecal tonsils at late stages of infection (21, 28, and 35 days). In general, the Mass strain was more frequently recovered from the tracheal and cecal tonsil tissues at earlier stages of infection and the Ark strain was recovered at later stages of infection.

Four genetic Mycoplasma gallisepticum (MG) polymerase chain reactions (PCRs) (16s rRNA PCR, three newly developed PCR methods that target surface protein genes [mgc2, LP (nested) and gapA (nested)]) were compared for analytical specificity and sensitivity and for diagnostic sensitivity (Se) and specificity of detection from tracheal swabs. The licensed MG DNA Test Kit Flock Chek test (IDEXX, Laboratories, Inc., Westbrook, ME) was as well evaluated for the diagnostic specificity and sensitivity of detection from tracheal swabs. Analytical specificity was evaluated for the four generic PCR methods using a panel of DNA samples from microorganisms that may be isolated from the trachea of commercial poultry and other fowl. PCR methods mgc2, nLP, and ngapA only amplified DNA from MG, whereas 16S rRNA PCR amplified DNA from MG and Mycoplasma imitans. The analytical sensitivity of the four generic PCR methods expressed in color-changing units (CCU)/amplification reaction was estimated for each PCR method and ranged from 4 to 400 CCU/reaction; the sensitivities of single PCR methods 16S rRNA and mgc2 were estimated at 40 CCU/reaction, the nLP at 400 CCU/reaction, and the ngapA at 4 CCU/reaction. The diagnostic sensitivity and specificity of MG detection from tracheal swab pools, as compared to isolation from choanal cleft swabs, was evaluated for the five PCR methods using three groups of birds exposed to vaccine strains ts-11 and 6/85 and to challenge strain R. All PCR methods were able to detect the vaccine strains and the challenge strain R directly from tracheal swabs, indicating that PCR primers from the different methods amplified divergent MG strains. Isolation and PCR results correlated satisfactorily among the three experimentally infected groups, with agreement values (k) ranging from 0.52 to 1.00. The ngapA, IDEXX, and mgc2 PCRs showed the best sensitivity (Se) ratios for detection of M. gallisepticum strains as compared to isolation. Compared to the ngapA and IDEXX PCR methods, the mgc2 PCR has a faster turnaround time, since this test consists of a single amplification reaction and the amplification product is detected by gel electrophoresis. Therefore, among the PCR methods evaluated in this study, the mgc2 PCR is the method of choice to further validate in the field.

Mycoplasma meleagridis (MM) is a known pathogen for turkeys only. In this study, MM was used to inoculate chicken embryos and tumor cells to assess its pathogenic potential for chickens. In chicken embryos, it caused abnormal-shaped toes and severely denuded tracheae. In chicken tumor cells, MM reduced the cellular capacity to release a chemoattractant that causes the migration of heterophils. MM also caused death and/or a reduced growth rate in chicken HD-11 cells, a macrophage-monocyte-derived cell line. Thus, the data show that MM is a potential pathogen for chicken embryos and chickens cells. Further exploration to determine the pathogenicity in chickens may be warranted.

In previous studies, the embryo lethality assay (ELA) discriminated between virulent and avirulent avian Escherichia coli isolates, and also proved to be highly correlated with mortality and morbidity results of the intravenous (IV) challenge model. In the current study, the same 20 avian E. coli isolates were used in subcutaneous (subQ) and intratracheal (IT) chicken challenge models in order to determine whether the results from the prior ELA challenges and/or the IV challenge model correlate with these models. The correlation observed between the two previous ELA trials and the combined mortality/morbidity percentages of the subQ challenge model were r = 0.792, P0.0001 for the first ELA trial and r = 0.738, P = 0.0002 for the second ELA trial. The IV challenge results were more highly correlated with the subQ challenge results (mortality/morbidity comparison, r = 0.894, P0.0001). The IV challenge mortality results were slightly correlated (r = 0.4810, P=0.0319) with the IT challenge results. Several of the isolates differed in their ability to produce mortality and/or morbidity with the different challenge models. The mortality/morbidity results of the IV and subQ challenges and the mortality results of the ELA were all positively correlated with the ability of an E. coli isolate to produce Colicin V (ColV) (r = 0.7131, P = 0.0004). The IT mortality results were slightly correlated with the production of ColV (r = 0.455, P = 0.049). The IT challenge results were only slightly correlated with resulting IV mortality and ColV production. Previous results indicate that the ELA correlates extremely well with the IV challenge model. The current study demonstrates that ELA also correlates well with the subQ challenge model. Overall, the conclusion of this study is that the ELA, IV, and subQ challenge models similarly demonstrate the ability to discriminate between virulent and avirulent avian E. coli isolates.

The objective of this research was to evaluate the safety of the 6/85 strain vaccine strain of Mycoplasma gallisepticum in turkeys by backpassing the vaccine strain up to 10 times by contact infection in turkeys and challenging turkeys with the resulting backpassaged strain. The vaccine strain, however, did not spread to in-contact turkeys, and it was necessary to reisolate the organism before challenging turkeys for the next passage. The challenge strain, therefore, was one that had been backpassaged four times in turkeys, with a total in vivo time in turkeys of 66 days. The backpassaged 6/85 vaccine strain was no different in pathogenicity than the original vaccine strain, except that at 10 days postchallenge, it was isolated in higher numbers from air sacs. Both the original 6/85 vaccine strain and the backpassaged strain were apathogenic in turkeys, except for a slightly increased diameter of the tracheal mucosa at 10 days postchallenge; at 20 days postchallenge the tracheal mucosal thickness was no different from that of controls.

The objective of this study was to determine the prevalence of avian influenza viruses (AIV) in bobwhite quail (Colinus virginianus) populations from the rolling plains of Texas, U. S. A. A total of 1320 swab samples (652 tracheal swabs and 668 cloacal swabs) and 44 serum samples were collected from wild-captured or hunter-harvested bobwhite quail from November 2009 to April 2011 at the Rolling Planes Quail Research Ranch, Fisher County, Texas, U. S. A. The presence of AIV in the swabs was determined by real-time reverse-transcription-PCR (rRT-PCR) and all samples positive or suspicious by rRT-PCR were further processed for virus isolation in embryonated chicken eggs. A total of 18 (1.4%) swab samples tested positive for AIV by rRT-PCR (cycle threshold [Ct] values35): 13 cloacal swabs (1.9%) and 5 tracheal swabs (0.8%). In addition, 100 (7.6%) swab samples were considered suspicious (Ct values 35.1-40): 69 cloacal swabs (10.3%) and 31 tracheal swabs (4.7%). No virus was isolated from any of the rRT-PCR-positive or suspicious samples tested. Additionally, 44 serum samples were screened for AIV antibodies and were negative. The results presented here indicate low prevalence of AIV in wild populations of bobwhite quail.

Infections of ostriches with avian influenza A viruses are generally associated with clinical disease, but the occasional high mortality in young birds does not appear to be related directly to virus pathotype. In this study we investigated the pathogenesis of two H7 viruses for 11-wk-old ostriches inoculated intranasally, and clinical symptoms, virus excretion, and immune response were studied. One of the viruses (A/Ostrich/Italy/1038/00) was highly pathogenic for chickens, whereas the other (A/Ostrich/South Africa/1609/91) was of low pathogenicity for chickens. Clinical signs in ostriches receiving virulent virus were slight depression and hemorrhagic diarrhea, while the group receiving avirulent virus was clinically normal except for green diarrhea. Both viruses were transmitted to in-contact sentinel birds housed with the infected groups 3 days postinfection. Postmortem examination of the birds infected (including the sentinel bird) with virus highly pathogenic for chickens were grossly normal except for localized pneumonic lesions. The results of the study are presented and discussed.

Avian infectious bronchitis virus (IBV) causes a highly contagious and economically significant disease in chickens. Establishment of a carrier state in IBV infection and the potential for the persistent virus to undergo mutations and recombination in chicken tissues have important consequences for disease management. Nevertheless, whether chickens can maintain persistent IBV infection in the absence of reinfection from exogenous sources or the presence of antibody in the host can modulate virus persistence remains unclear. Indeed, whether or not IBV genome can undergo genetic changes during in vivo infection has not been demonstrated experimentally. In the present study, IBV shedding and tissue persistence were monitored in individual chickens maintained under strict isolation that precluded reinfection from exogenous sources. In the first of two experiments, intranasal exposure of 6-wk-old antibody-free chickens to IBV vaccine virus resulted in intermittent shedding of the virus from both trachea and cloaca of individual birds for up to 63 days. Also, the virus was recovered from the internal organs (spleen, gonad, kidney, lung, cecal tonsil, and cloacal bursa) of six of eight birds killed at various intervals between 27 and 163 days postinoculation (DPI). In the second experiment, IBV exposure of 1-day-old maternal antibody-positive chicks led to periodic virus shedding from the trachea and cloaca in all chickens until 77 days; however, internal organs (lungs and kidneys) of only one of seven birds (killed at 175 DPI) were virus positive, suggesting that presence of antibody at the time of infection protects internal organs from IBV infection. When the lung and kidney isolates of IBV from the latter experiment were compared with the parent-vaccine virus, no changes in their antigenicity, tissue tropism, or the nucleotide sequence of the S1 glycoprotein gene were observed. These findings indicate that, unlike the mammalian coronaviruses, propensity for frequent genetic change may not be inherent in the IBV genome.

The efficacy of four different commercial live vaccines (vaccines A, B, C, and D) against the infectious laryngotracheitis virus (ILTV) was assessed in specific-pathogen-free (SPF) chickens. SPF chickens were vaccinated intraocularly at 6 wk old with ILTV live vaccines and were challenged intratracheally with the N91B01 strain of virulent Korean ILTV 2 wk after vaccination. The immunity against ILTV live vaccines was assessed by the incidence of latent infection by the challenge virus in the chickens' tracheas and trigeminal ganglia, the reisolation rate of the challenge virus, and the clinical signs in the chickens challenged with the N91B01 strain of ILTV. The latent infection in chickens was assessed by nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Our data showed that the clinical signs and challenge virus isolation were negative in all chickens receiving four difference commercial ILTV live vaccines. The viral DNA of the vaccine strain, but not that of the challenge virus, was detected in chickens vaccinated with vaccine A by nested PCR-RFLP. The viral DNAs of both the vaccine and challenge strains were detected from chickens vaccinated with vaccines B, C, and D. This study showed that only vaccine A can protect chickens from latent infection with the field virulent ILTV. We speculate that the efficacy of infectious laryngotracheitis live vaccines to protect chickens from latent infection with virulent ILTVs can be assessed by nested PCR-RFLP analysis.

The live attenuated temperature-sensitive (ts+) Mycoplasma synovia (MS) strain, MS-H, is used as a vaccine in a number of countries to control virulent MS infection in commercial chicken flocks. Nine out of 50 isolates made from flocks vaccinated with MS-H were found to have lost the ts+ phenotype of the original vaccine strain. In order to examine the influence of the ts- phenotype on virulence of the isolates, four of the ts- isolates, the MS-H vaccine, and the vaccine parent strain 86079/7NS were administered by aerosol in conjunction with infectious bronchitis virus to 3-wk-old specific-pathogen-free chickens. The four ts- clones induced only minimal air sac lesions that were not different in severity from those caused by MS-H vaccine; however, the vaccine parent strain 86079/7NS caused air sac lesions that were significantly greater than those of MS-H and all ts- clones. The vaccine parent strain 86079/7NS and two of the ts- clones were recovered from the air sacs of the respectively infected chickens whereas the MS-H vaccine and two other ts- clones were not. Three of the ts- isolates caused increased tracheal mucosal thicknesses that were significantly greater than those from birds inoculated with MS-H, and one caused increased tracheal mucosal thicknesses that were significantly less than those from birds inoculated with 86079/7NS. In conclusion, unlike the MS-H vaccine, the MS-H ts- clones were associated with minor changes in tracheal mucosa; however, unlike the vaccine parent strain, they did not induce lesions in the air sacs. These results suggest that factors other than ts+ phenotype are involved in the attenuation of the MS-H vaccine.

SUMMARY. In serum, tracheal wash fluid, and bile from chickens that were inoculated with live or inactivated Newcastle disease virus (NDV), the kinetics and immunoglobulin (Ig) class distribution of an antibody response were demonstrated. The Ig classes (IgM, IgG, and IgA) were captured using monoclonal antibodies (MAbs) in enzyme-linked immunosorbent assays (Ig-capture ELISA). The antibody specificity of the captured Ig was confirmed by binding of NDV. After inoculation with live virus, antibodies of the IgG and IgM classes were mainly found in serum. IgM was produced early from day 4 postexposure (PE) onward, IgG was detected later from day 7 PE onward, and in the tracheal wash fluid and bile, all three Ig classes were demonstrated. After inoculation of inactivated virus, a delayed response of all three classes was observed in serum, and only IgM and IgG were recognized in the tracheal fluid and bile. The type of vaccine and the route of antigen entrance may have determined the immunoglobulin class produced. The Ig-capture ELISA assay developed in this study can be useful for evaluating various strategies to improve the efficacy of Newcastle disease vaccines and to study the evoked immune mechanisms.

Thirty-eight cases were identified in which a nonfermentative, gram-negative, rod-shaped bacterium was isolated from the respiratory system of turkeys and chickens. Cases were submitted from various parts of the country. Preliminary assessment of phenotypic characteristics indicated this bacterium was different from common pathogenic or opportunistic bacteria isolated from the avian respiratory tract. Most cases reported a history of respiratory distress and/or increased flock mortality. Lesions seen in infected birds included tracheitis and pneumonia, which correlate with the sites of isolation. Sixty-one percent of the isolations were made from the trachea and 25% from the lung. Age of infected birds ranged from 35 to 315 days in turkeys and 53 days to 3 yr in chickens. In most instances (90%), other bacteria were also isolated from affected sites. The significance of this organism in respiratory disease in birds is unknown.

During 2007, two outbreaks of avian influenza virus (AIV) in backyard and commercial ostrich flocks were first reported in the Kingdom of Saudi Arabia (KSA). The infected ostriches suffered from depression, anorexia, and diarrhea and some exhibited sudden death. A rapid AIV-group antigen detection and real-time reverse-transcription PCR (rtRT-PCR) were initially performed on cloacal and tracheal swabs collected from diseased birds. Pools from positive-tested swabs for each flock were utilized for virus isolation in specific-pathogen-free embryonating chicken eggs. H5N1 AIV was identified in the harvested allantoic fluids by hemagglutination followed by hemagglutination inhibition and rtRT-PCR. The viruses responsible for these two outbreaks were sequenced and characterized as HPAIV H5N1 (A/ostrich/Saudi Arabia/6732-3/2007 and A/ostrich/Saudi Arabia/3489-73VIR08/ 2007) from backyard and commercial flocks, respectively. Phylogenetic analysis of both isolates revealed that the two viruses belong to clade 2.2 sublineage II and cluster with the HPAIV H5N1 isolated from falcons and turkeys during 2007 in KSA.

Recent phylogenetic studies have identified different genotypic lineages of infectious laryngotracheitis virus (ILTV), and these lineages can recombine in the field. The emergence of virulent recombinant field strains of ILTV by natural recombination between commercial vaccines belonging to different genotypic lineages has been reported recently. Despite the use of attenuated ILTV vaccines, these recombinant viruses were able to spread and cause disease in commercial poultry flocks, raising the question of whether the different lineages of ILTV can induce cross-protective immune responses. This study examined the capacity of the Australian-origin A20 ILTV vaccine to protect against challenge with the class 8 ILTV recombinant virus, the genome of which is predominantly derived from a heterologous genotypic lineage. Following challenge, birds vaccinated via eyedrop were protected from clinical signs of disease and pathological changes in the tracheal mucosa, although they were not completely protected from viral infection or replication. In contrast, the challenge virus induced severe clinical signs and tracheal pathology in unvaccinated birds. This is the first study to examine the ability of a vaccine from the Australian lineage to protect against challenge with a virus from a heterologous lineage. These results suggest that the two distinct genotypic lineages of ILTV can both induce cross-protection, indicating that current commercial vaccines are still likely to assist in control of ILTV in the poultry industry, in spite of the emergence of novel recombinants derived from different genotypic lineages.

Newcastle disease (ND) is prevalent worldwide and causes significant clinical and economic losses to the poultry industry. Current vaccine programs using live attenuated vaccines and inactivated vaccines have limitations, and new vaccines with distinct features are needed. To offer an alternative solution to control ND, a turkey herpesvirus vector Newcastle disease vaccine (HVT/ND) expressing the fusion gene of Newcastle disease virus (NDV) has been developed. First, immunogenicity of the HVT/ND was evaluated in specific-pathogen-free layer chickens after vaccination by the in ovo route to 18-day-old embryos or by the subcutaneous route to 1-day-old chicks. Antibodies against NDV were detected at 24 days of age using a commercial NDV enzyme-linked immunosorbent assay (ELISA) kit and the hemagglutination inhibition test. At least 90% of chickens were protected against challenge with velogenic neurotropic NDV Texas GB strain (genotype II; pathotype velogenic) at 4 wk of age, while none of the nonvaccinated, challenged controls were protected from challenge. Second, the age at which a vaccinated chicken elicits an immunologic response to the HVT/ND prepared for this study, and thus is protected from ND virus, was assessed in commercial broiler chickens after in ovo vaccination of 18-day-old embryos. Challenge was conducted using a low-virulence NDV strain (genotype II; pathotype lentogenic) via the respiratory tract each week between 1 and 5 wk of age, in order to mimic the situation in areas where virulent NDV strains do not normally exist and low-virulence strains cause mild respiratory symptoms leading to economic losses. Protection was evaluated by the presence or absence of isolated virus from tracheal swabs at 5 days postchallenge. Partial protection was observed at 3 wk of age, when 6 out of 10 (60%) chickens were protected. Full protection was obtained at 4 and 5 wk of age, when 9 out of 10 (90%) and 10 out of 10 (100%) chickens were protected, respectively. Finally, protection against challenge with virulent Texas GB strain at 19 wk of age was evaluated in commercial female layer chickens vaccinated at 1 day of age with HVT/ND. All of the vaccinated chickens were protected, while all of the challenge controls succumbed to the challenge. Furthermore, anti-NDV antibodies measured by ELISA were maintained through 50 wk of age.

Several epidemiologic surveillance studies have implicated backyard flocks as a reservoir for poultry diseases; however, much debate still exists over the risk these small flocks pose. To evaluate this concern, the prevalence of Newcastle disease (ND), infectious laryngotracheitis (ILT), Mycoplasma gallisepticum (MG), and Salmonella was determined in 39 Maryland backyard flocks. Serum, tracheal, and cloacal swabs were randomly collected from 262 birds throughout nine counties in Maryland. Through PCR and ELISA analysis, disease prevalence and seroprevalence were determined in flocks, respectively, for the following: ND (0%, 23%); ILT (26%, 77%); MG (3%, 13%); and Salmonella (0%, not done). Vaccine status could not be accurately confirmed. Premise positives were further differentiated and identified by partial nucleotide sequencing. Screening of the 10 ILT premise positives showed that most were live attenuated vaccines: eight matched a tissue culture origin vaccine, one matched a chicken embryo origin (CEO) vaccine, and one was CEO related. The single MG-positive flock, also positive for the CEO-related sequence, was identified as the infectious S6 strain. The prevalence rates for these economically important poultry diseases ranged from none to relatively low, with the vast majority of sampled flocks presenting no clinical signs.

SUMMARY. Tracheas from chickens infected both in the field and experimentally with lentogenic Newcastle disease virus (also known as avian paramyxovirus-1 [APMV-1] and referred to here as “lentogenic NDV”) were examined histopathologically to score degree of pathologic changes and by immunohistochemistry to determine presence of viral protein. In the field cases there was often a striking lack of correlation between severity of tracheal lesions and amount of immunohistochemical signal for APMV-1 protein. Experimental cases had minimal pathologic changes and also minimal immunohistochemical signal. Positive cells were often associated with surface deciliation. It may be that lentogenic NDV has only a minor role as a respiratory pathogen, merely compromising the mucosa to allow other respiratory pathogens to infect and worsen the clinical and pathologic presentation.

Egyptian geese (Alopochen aegypticus), a duck species endemic to sub-Saharan Africa and occasionally implicated in the transmission of avian influenza viruses (AIV) to farmed ostriches, were experimentally infected with low pathogenicity H7N1 and H6N8 viruses to assess viral shedding and immune profiles. Following the first infection with H7N1 virus, high titers of virus were shed from both the tracheae and cloacae for at least 7 days postinfection, and tracheal shedding lasting until day 14. All detectable shedding from both tracheae and cloacae had ceased within 28 days of infection. Antibody titers peaked at day 7 postinfection, but the initial immune response was short-lived. Birds that received a second challenge with the homologous H7N1 virus mounted a more robust response that lasted beyond 66 days postchallenge, and H7N1 virus was detected, albeit at much lower levels, until day 28 post secondary infection (psi) in the cloaca and beyond day 28 psi in the trachea. Birds that received an initial infection with H7N1 virus were also challenged with H6N8 virus, and because a comparable shedding pattern to the H7N1 challenge group was observed, we concluded that the effect of any nonspecific immunity was negligible.

SUMMARY. We evaluated the pathogenicity of three live Mycoplasma gallisepticum (MG) vaccine candidates by infection via aerosol of 3-wk-old chickens with log phase broth cultures (trial 1). Two of the candidates (K3020 and K4649A) colonized only 10% and 20% of the chickens, respectively, unlike K2101 (K-strain), which was reisolated from all of the vaccinated chickens tested. K-strain inoculation did not result in significant air sac or tracheal lesions in chickens at 10 and 39 days postinfection (P # 0.05). The efficacy of K-strain as a live vaccine was evaluated in trial 2, by challenge of vaccinated chickens with virulent R-strain via aerosol at 6 wk postvaccination. K-strain vaccination resulted in significant protection from air sac and tracheal lesions (P # 0.05). The K-strain was further investigated to evaluate transmissibility (trial 3), colonization and persistence of infection following aerosol administration (trial 4), genetic and phenotypic stability following back passage through chickens (trial 5), and vertical transmission (trial 6). The K-strain had a low rate of horizontal transmission; it remained primarily in the respiratory system of inoculated birds and persisted in the upper respiratory tract for the duration of the trial 4 (5 mo). There was no increase in virulence of K-strain when it was back passaged five times through chickens, and no vertical transmission of K-strain was detected. K-strain showed great potential as a safe and effective live MG vaccine.

SUMMARY. Tracheal and cloacal swabs as well as blood samples from 408 feral urban (Columba livia forma domestica) and 170 free-ranging wood pigeons (Columba palumbus) in Germany were tested for infection with avian influenza viruses (AIVs). Neither influenza A virus was isolated in the swab samples nor influenza A virus RNA detected using real-time reverse transcriptase–PCR (RT-qPCR). In three urban feral pigeons, avian paramyxovirus (APMV) serotype 1 was isolated. Two of 123 serum samples from hunted free-ranging wood pigeons contained specific antibodies against influenza A virus but not against the subtypes H5 and H7. In conclusion, our study indicates that even after the occurrence of zoonotic highly pathogenic avian influenza virus (HPAIV) subtype H5N1 in the area of investigation in Germany, pigeons do not play a major part in the transmission of influenza viruses. The risk of AIV infection for humans from urban and free-ranging wood pigeons is, if at all, of minimal importance.

To assess the susceptibility of pigeons (Columba livia) to infection with H5N1 high pathogenicity avian influenza virus (HPAIV), four groups of 1-yr-old and 4-wk-old racing pigeons (10 birds in each group) were inoculated oculonasally with 106 50% egg infectious dose (EID50) of A/crested eagle/Belgium/01/2004 (clade 1) or A/swan/Poland/305-135V08/2006 (clade 2.2). Contact specific-pathogen-free (SPF) chickens were kept in the same isolators as young pigeons (two chickens per group). At 3, 5, 7, 10, and 14 days postinfection (PI) two pigeons from each infected group were selected randomly, and oropharyngeal and cloacal swabs (pigeons and contact chickens) as well as a number of internal organs (pigeons) were collected for viral RNA detection in real-time reverse transcription PCR (RRT-PCR) and histopathology. At the end of the experiment (14 days PI) blood samples from two pigeons in each group and from contact SPF chickens were also collected, and sera were tested using hemagglutination inhibition (HI) test and blocking enzyme-linked immunosorbent assay (bELISA). During the observation period all pigeons remained clinically healthy, and no gross lesions were observed in any of the infected groups. SPF contact chickens were also healthy and negative in RRT-PCR and HI tests. However, the clade 1 H5N1 virus produced more sustained infection manifested by the presence of histopathologic changes (consisting mainly of mild to moderate hemorrhagic and inflammatory lesions), prolonged persistence of viral RNA (detectable between 3 and 10 days PI) in a variety of tissues of both adult and juvenile birds (with highest RNA load in lungs and brain) as well as slight viral shedding from the trachea and cloaca, but without transmission to SPF contact chickens. Additionally, two clade 1-infected adult pigeons sacrificed at the end of experiment showed seroconversion in bELISA and HI test (using homologous virus as antigen). The viral RNA was found only at day 3 PI in one adult pigeon inoculated with dade 2.2 H5N1 virus, but neither microscopic lesions nor seroconversion were found in any other tested birds inoculated with A/swan/Poland/305-135V08/2006. Our results support the observations that pigeons are resistant to H5N1 HPAIV (no deaths or clinical signs), but there may be clade-dependent differences in the pathogenic potentials of H5N1 HPAIV of Asian origin.

Arkansas (Ark)-type infectious bronchitis virus (IBV) subpopulations with an S gene sequence distinct from the vaccine predominant consensus were previously found in the upper respiratory tract of chickens within 3 days after inoculation. This finding indicated that a distinct virus subpopulation was rapidly positively selected by the chicken upper respiratory tract. We hypothesized that during host invasion, the replicating IBV population further changes as it confronts the distinct environments of different tissues, leading to selection of the most fit population. We inoculated 15-day-old chickens with 10(4) 50% embryo infective doses of an Ark-type IBV commercial vaccine via the ocular and nasal routes and characterized the sequences of the S1 gene of IBV contained in tear fluid, trachea, and reproductive tract of individual chickens at different times postinoculation. The predominant IBV phenotype contained in the vaccine (before inoculation) became a minor or nondetectable population at all times in all tissues after replication in the majority of the chickens, corroborating our previous findings. Five new predominant populations designated component (C) 1 through C5, showing distinct nonsynonymous changes, i.e., nucleotide changes resulting in different amino acids encoded and thus in a phenotypic change of the predominant virus population, were detected in the tissues or fluids of individual vaccinated chickens. Due to the different biochemical properties of some amino acids that changed in the S1 glycoprotein, we anticipate that phenotypic shift occurred during the invasion process. Significant differences were detected in the incidence of some distinct IBV predominant populations in tissues and fluids; e.g., phenotype C1 showed the highest incidence in the reproductive tract of the chickens, achieving a significant difference versus its incidence in the trachea (P0.05). These results indicate for the first time that IBV undergoes intraspatial variation during host invasion, i.e., the dominant genotype/phenotype further changes during host invasion as the microenvironment of distinct tissues exerts selective pressure on the replicating virus population.

Bordetella hinzii is commonly acquired from the respiratory tract of diseased poultry but is generally regarded as nonpathogenic in avian hosts because attempts to demonstrate disease following experimental infection of chickens and turkeys have failed. Recently, with the availability of highly specific DNA-based methods for identification of this agent, it was recognized that some isolates used in previous studies were misidentified at the time of their acquisition as Bordetella avium, B. avium-like, or Alcaligenes faecalis type II, including a subset reported to cause disease in turkey poults. In this study six strains of B. hinzii, genetically distinct and representing all known host species, were evaluated for their ability to colonize and cause disease in turkeys following intranasal administration. Although five strains were able to colonize the tracheas of turkey poults, only a subset induced clinical signs of disease, B. hinzii-specific antibodies, or tracheal lesions. The sixth isolate was undetectable in tracheal swabs obtained 1 or 2 weeks postinfection. Birds of this group displayed no clinical signs and minimal tracheal lesions. All remained B. hinzii seronegative. Three of the six strains, differing in their capacity to colonize and/or cause disease in turkeys, were used to infect chicks intranasally. Only one was able to colonize the trachea but did not induce tracheal lesions. No clinical signs of disease were observed in any chick. These results demonstrate that some strains of B. hinzii are virulent in turkey poults and may asymptomatically colonize chicks, and suggest this agent may be of concern to poultry producers.

The Asian H5N1 highly pathogenic avian influenza (HPAI) virus causes a systemic disease with high mortality of poultry and is potentially zoonotic. In both chickens and ducks, the virus has been demonstrated to replicate in both cardiac and skeletal muscle cells. Experimentally, H5N1 HPAI virus has been transmitted to chickens through the consumption of raw infected meat. In this study, we investigated virus replication in cardiac and skeletal muscle and in the trachea of chickens after experimental intranasal inoculation with the H5N1 HPAI virus. The virus was detected in tissues by real-time reverse transcription-polymerase chain reaction (RRT-PCR) and virus isolation, and in the trachea by RRT-PCR and a commercial avian influenza (AI) viral antigen detection test. A modified RNA extraction protocol was developed for rapid detection of the virus in tissues by RRT-PCR. The H5N1 HPAI virus was sporadically detected in meat and the tracheas of infected birds without any clinical sign of disease as early as 6 hr postinfection (PI), and was detected in all samples tested at 24 hr PI and later. No differences in sensitivity were seen between virus isolation and RRT-PCR in meat samples. The AI viral antigen detection test on tracheal swabs was a useful method for identifying infected chickens when they were sick or dead, but was less sensitive in detecting infected birds when they were preclinical. This study provides data indicating that preslaughter tracheal swab testing can identify birds infected with HPAI among the daily mortality and prevent infected flocks from being sent to processing plants. In addition, the modified RNA extraction and RRT-PCR test on meat samples provide a rapid and sensitive method of identifying HPAI virus in illegal contraband or domestic meat samples.

SUMMARY. Studies were performed to determine if mucosal vaccination with inactivated avian metapneumovirus (aMPV) subtype C protected turkey poults from clinical disease and virus replication following mucosal challenge. Decreases in clinical disease were not observed in vaccinated groups, and the vaccine failed to inhibit virus replication in the tracheas of 96% of vaccinated birds. Histopathologically, enhancement of pulmonary lesions following virus challenge was associated with birds receiving the inactivated aMPV vaccine compared to unvaccinated birds. As determined by an enzyme-linked immunosorbent assay (ELISA), all virus-challenged groups increased serum immunoglobulin (Ig) G and IgA antibody production against the virus following challenge; however, the unvaccinated aMPV-challenged group displayed the highest increases in virus-neutralizing antibody. On the basis of these results it is concluded that intranasal vaccination with inactivated aMPV does not induce protective immunity, reduce virus shedding, or result in decreased histopathologic lesions.

The onset of protective immunity with MS-H was determined through experimental challenge and compared with the parent strain 86079/7NS. MS-H vaccinates and 86079/7NS inoculates were challenged at 1, 2, 3, 4, 5, and 6 wk after vaccination, then examined 2 wk after challenge for signs of respiratory disease. Serologic results indicated that 100% of MS-H vaccinates had antibodies to MS by 3 wk after vaccination and 100% of 86079/7NS inoculates were positive by 2 wk after inoculation. From 3 wk after vaccination, MS-H vaccinates had a significantly lower incidence of air sac lesions and, from 4 wk after vaccination, a significantly lower air sac lesion severity. In 86079/7NS-inoculated birds, a significantly lower incidence of air sac lesions was observed from 1 wk after inoculation, and air sac lesion severity was significantly lower than the unvaccinated controls at 3 wk after inoculation. It would appear that, under the conditions of this experiment, protective immunity elicited by MS-H appeared at 4 wk after vaccination, slightly later than the appearance of serum antibody. Although the MS-H vaccine was slower to establish protective immunity than 86079/7NS, there was no significant difference between the two strains by 4 wk after vaccination or inoculation.

Low-pathogenicity avian influenza (LPAI) subtype H7N3 was diagnosed on a two-age broiler breeder farm in Abbotsford, British Columbia (BC), in early February 2004. The presenting complaint in the older index flock was feed refusal, with 0.5% mortality over 72 hr that resolved over the following week Ten days after the initial complaint in the index flock, a younger flock in an adjacent barn experienced an abrupt spike in mortality (25% in 48 hr). The gross lesions of tracheal hyperemia and hilar pulmonary consolidation were subtle and nonspecific, and the diagnosis of avian influenza required laboratory confirmation. Two different viruses were isolated from the index farm: a LPAI (H7N3) was isolated from the older flock and a high-pathogenicity avian influenza (HPAI) (H7N3), which had an additional 21 base insertion at the hemagglutinin-cleavage site, was isolated from the younger flock. The presence of this insertion sequence and the similarity of adjacent sequences indicate that the LPAI had mutated into HPAI at some point between the first and second barn. Despite enhanced on-farm biosecurity measures, the virus was not contained on the index farm and eventually spread to over 40 commercial poultry facilities before massive depopulation efforts enabled its eradication.