Infectious Bronchitis Virus Immune Responses in the Harderian Gland upon Initial Vaccination.

In recent years, Arkansas Delmarva Poultry Industry (ArkDPI)-derived infectious bronchitis (IB) virus (IBV) vaccines have been used to characterize the immune responses of chickens subsequent to vaccination on day of hatch or beyond. Perhaps because ArkDPI vaccines display increased heterogeneity, the results on cell immune responses have shown ambiguity. In the current study, we investigated the effects of vaccination with a highly stable and homogeneous Massachusetts (Mass)-type vaccine on days 1 or 7 of age on Harderian gland (HG) responses. Confirming previous studies, both IBV serum antibodies and lachrymal IgA levels were greater upon vaccination on day 7 compared with vaccination on day 1 of age. Unlike results with ArkDPI viruses, a clear trend was detected for both B and T cells in the HG after Mass-type vaccination. Consistent with antibody responses, B- and T-helper (CD3+CD4+) cell frequencies were higher in birds vaccinated on day 7 of age. Cytotoxic T cells (CD3+CD8+) were also increased compared with chickens vaccinated on day 1 of age. Depending on the most likely age of IB outbreaks to occur in a particular region, postponing the first IBV vaccination may optimize immune responses.

Nota de Investigación- Respuestas inmunes al virus de la bronquitis infecciosa en la glándula de Harder después de una vacunación inicial En los últimos años, las vacunas contra el virus de la bronquitis infecciosa (IB) derivadas de la cepa Arkansas Industria Avícola de Delmarva (ArkDPI) se han utilizado para caracterizar las respuestas inmunes de los pollos después de la vacunación en el día de la eclosión o en días posteriores. Posiblemente debido a que las vacunas ArkDPI muestran una mayor heterogeneidad, los resultados sobre las respuestas inmunes celulares han mostrado ambigüedad. En el presente estudio, se investigaron los efectos de la vacunación con una vacuna de tipo Massachusetts (Mass) que es altamente estable y homogénea en los días uno o siete de edad con relación a las respuestas en la glándula de Harder (HG). Confirmando estudios previos, tanto los anticuerpos séricos contra el virus de la bronquitis infecciosa como los niveles de IgA lacrimal fueron mayores tras la vacunación al día siete en comparación con la vacunación al primer día de edad. A diferencia de los resultados con los virus ArkDPI, se detectó una clara tendencia para las células B y T en la glándula de Harder después de la vacunación con el tipo Mass. De acuerdo con las respuestas de anticuerpos, las frecuencias de células B y T cooperadoras (CD3+ CD4+) fueron más altas en las aves vacunadas en el día siete de edad. Las células T citotóxicas (CD3+CD8+) también aumentaron en comparación con los pollos vacunados en el primer día de edad. Dependiendo de la edad más probable en que ocurran brotes por bronquitis infecciosa en una región en particular, posponer la primera vacuna contra el IBV puede optimizar las respuestas inmunes.

Ocular exposure to infectious laryngotracheitis virus alters leukocyte subsets in the head-associated lymphoid tissues and trachea of 6-week-old White Leghorn chickens.

Infectious laryngotracheitis virus (ILTV), an alphaherpesvirus, causes acute respiratory disease primarily infecting the upper respiratory tract and conjunctiva. Administration of live attenuated ILTV vaccines via eye drop, drinking water, or by coarse spray elicits protective mucosal immunity in the head-associated lymphoid tissues (HALT), of which conjunctiva-associated lymphoid tissue (CALT) and the Harderian gland (HG) are important tissue components. The trachea, a non-lymphoid tissue, also receives significant influx of inflammatory cells that dictate the outcome of ILTV infection. The objective of this study was to evaluate leukocyte cellular and phenotypic changes in the CALT, HG and trachea following ocular infection with a virulent ILTV strain. At 1, 3, 5, 7 and 9 days post-infection, CALT, HG, and trachea of 6-week-old specific pathogen free (SPF) chickens ocularly-exposed to vehicle or virulent ILTV strain 63140 were dissociated, the cells enumerated and then phenotyped using flow cytometry. The CALT had the highest viral genomic load, which peaked on day 3. In ILTV-infected birds, the CALT had a decreased percentage of leukocytes. This was reflected by decreased numbers of MHCI+MHCII-, MHCI+MHCIIlow+, and CD4+ cells, while IgM+ and MHCI+MHCIIHigh+ expressing cell populations increased. In the HG, the most notable change in cells from ILTV-infected birds was a decrease in IgM expressing cells and histologically, an increase in Mott cells. In summary, an acute, ocular exposure to ILTV strain 63140 in young birds shifts subsets of lymphocyte populations in the CALT and HG with minimal impact on the trachea.

Novel insights into the host immune response of chicken Harderian gland tissue during Newcastle disease virus infection and heat treatment.

BACKGROUND: Newcastle disease virus, in its most pathogenic form, threatens the livelihood of rural poultry farmers where there is a limited infrastructure and service for vaccinations to prevent outbreaks of the virus. Previously reported studies on the host response to Newcastle disease in chickens have not examined the disease under abiotic stressors, such as heat, which commonly experienced by chickens in regions such as Africa. The objective of this study was to elucidate the underlying biological mechanisms that contribute to disease resistance in chickens to the Newcastle disease virus while under the effects of heat stress. RESULTS: Differential gene expression analysis identified genes differentially expressed between treated and non-treated birds across three time points (2, 6, and 10 days post-infection) in Fayoumi and Leghorn birds. Across the three time points, Fayoumi had very few genes differentially expressed between treated and non-treated groups at 2 and 6 days post-infection. However, 202 genes were differentially expressed at 10 days post-infection. Alternatively, Leghorn had very few genes differentially expressed at 2 and 10 days post-infection but had 167 differentially expressed genes at 6 days post-infection. Very few differentially expressed genes were shared between the two genetic lines, and pathway analysis found unique signaling pathways specific to each genetic line. Fayoumi had significantly lower viral load, higher viral clearance, higher anti-NDV antibody levels, and fewer viral transcripts detected compared to Leghorns. Fayoumis activated immune related pathways including SAPK/JNK and p38 MAPK signaling pathways at earlier time points, while Leghorn would activate these same pathways at a later time. Further analysis revealed activation of the GP6 signaling pathway that may be responsible for the susceptible Leghorn response. CONCLUSIONS: The findings in this study confirmed our hypothesis that the Fayoumi line was more resistant to Newcastle disease virus infection compared to the Leghorn line. Within line and interaction analysis demonstrated substantial differences in response patterns between the two genetic lines that was not observed from the within line contrasts. This study has provided novel insights into the transcriptome response of the Harderian gland tissue during Newcastle disease virus infection while under heat stress utilizing a unique resistant and susceptible model.

Cytokine gene transcription in the trachea, Harderian gland, and trigeminal ganglia of chickens inoculated with virulent infectious laryngotracheitis virus (ILTV) strain.

The objective of this study was to determine how cytokine transcription profiles correlate with patterns of infectious laryngotracheitis virus (ILTV) replication in the trachea, Harderian gland, and trigeminal ganglia during the early and late stages of infection after intratracheal inoculation. Viral genomes and transcripts were detected in the trachea and Harderian gland but not in trigeminal ganglia. The onset of viral replication in the trachea was detected at day one post-infection and peaked by day three post-infection. The peak of pro-inflammatory (CXCLi2, IL-1ß, IFN-γ) and anti-inflammatory (IL-13, IL-10) cytokine gene transcription, 5 days post-infection, coincided with the increased recruitment of inflammatory cells, extensive tissue damage, and limiting of virus replication in the trachea. In contrast, transcription of the IFN-ß gene in the trachea remained unaffected suggesting that ILTV infection blocks type I interferon responses. In the Harderian gland, the most evident transcription change was the early and transient upregulation of the IFN-γ gene at 1 day post-infection, which suggests that the Harderian gland is prepared to rapidly respond to ILTV infection. Overall, results from this study suggest that regulation of Th1 effector cells and macrophage activity by Th1/2 cytokines was pertinent to maintain a balanced immune response capable of providing an adequate Th1-mediated protective immunity, while sustaining some immune homeostasis in preparation for the regeneration of the tracheal mucosa.

Novel analysis of the Harderian gland transcriptome response to Newcastle disease virus in two inbred chicken lines.

Behind each eye of the chicken resides a unique lymph tissue, the Harderian gland, for which RNA sequencing (RNA-seq) analysis is novel. We characterized the response of this tissue to Newcastle disease virus (NDV) in two inbred lines with different susceptibility to NDV across three time points. Three-week-old relatively resistant (Fayoumi) and relatively susceptible (Leghorn) birds were inoculated with a high-titered (107EID50) La Sota strain of NDV via an oculonasal route. At 2, 6, and 10 days post infection (dpi) Harderian glands were collected and analyzed via RNA-seq. The Fayoumi had significantly more detectable viral transcripts in the Harderian gland at 2 dpi than the Leghorn, but cleared the virus by 6 dpi. At all three time points, few genes were declared differentially expressed (DE) between the challenged and nonchallenged birds, except for the Leghorns at 6 dpi, and these DE genes were predicted to activate an adaptive immune response. Relative to the Leghorn, the Fayoumi was predicted to activate more immune pathways in both challenged and nonchallenged birds suggesting a more elevated immune system in the Fayoumis under homeostatic conditions. Overall, this study helped characterize the function of this important tissue and its response to NDV.

The proportion of specific viral subpopulations in attenuated Arkansas Delmarva poultry industry infectious bronchitis vaccines influences vaccination outcome.

We investigated the significance of differing proportions of specific subpopulations among commercial Arkansas (Ark) Delmarva poultry industry (DPI) vaccines with regard to vaccination outcome. Two ArkDPI-derived vaccines that contain a higher proportion of viruses with S1 genes that become selected during replication in chickens exhibited more rapid establishment of those selected subpopulations in chickens, produced significantly higher viral loads in tears, and induced higher antibody responses compared with two other ArkDPI vaccines with lower proportions of viruses that become selected in chickens. The presence of higher proportions of selected subpopulations was also associated with a significantly higher incidence of respiratory signs early after vaccination and in some cases more severe tracheal lesions. However, one of the ArkDPI-derived vaccines with a lower proportion of selected subpopulations, despite producing a lower viral load in tears, also induced a higher incidence of respiratory signs later after vaccination and more severe tracheal lesions. Furthermore, one of the ArkDPI-derived vaccines with a higher proportion of selected subpopulations, despite producing a higher viral loads in tears, resulted in less severe tracheal damage. These discrepancies suggest that infectious bronchitis virus (IBV) load in tears may not always predict degree of tracheal damage and that phenotypic characteristics other than S1 may also be involved in severity of vaccine reactions following ArkDPI vaccine administration. We observed lower antibody responses to the vaccines that produced lower viral loads, which might contribute to the persistence of Ark serotype IBV vaccines observed in commercial flocks.

Local and systemic immune responses following infection of broiler-type chickens with avian Metapneumovirus subtypes A and B.

Infections with avian Metapneumovirus (aMPV) are often associated with swollen head syndrome in meat type chickens. Previous studies in turkeys have demonstrated that local humoral and cell-mediated immunity plays a role in aMPV-infection. Previous experimental and field observations indicated that the susceptibility of broilers and their immune reactions to aMPV may differ from turkeys. In the presented study local and systemic immune reactions of broilers were investigated after experimental infections with subtypes A and B aMPV of turkey origin. Both virus subtypes induced a mild respiratory disease. The recovery from respiratory signs correlated with the induction of local and systemic aMPV virus-neutralizing antibodies, which began to rise at 6 days post infection (dpi), when the peak of clinical signs was observed. In a different manner to the virus neutralizing (VN) and IgG-ELISA serum antibody titres, which showed high levels until the end of the experiments between 24 and 28 dpi, the specific IgA-ELISA and VN-antibody levels in tracheal washes decreased by 10 and 14 dpi, respectively, which may explain the recurring aMPV-infections in the field. Ex vivo cultured spleen cells from aMPV-infected broilers released at 3 and 6 dpi higher levels of IFN-γ after stimulation with Concanavalin A as compared to virus-free birds. In agreement with studies in turkeys, aMPV-infected broilers showed a clear CD4+ T cell accumulation in the Harderian gland (HG) at 6 dpi (P<0.05). In contrast to other investigations in turkeys aMPV-infected broilers showed an increase in the number of CD8alpha+ cells at 6 dpi compared to virus-free birds (P<0.05). The numbers of local B cells in the Harderian gland were not affected by the infection. Both aMPV A and B induced up-regulation of interferon (IFN)-γ mRNA-expression in the nasal turbinates, while in the Harderian gland only aMPV-A induced enhanced IFN-γ expression at 3 dpi. The differences in systemic and local T cell and possibly natural killer cell activity in the HG between turkeys and chickens may explain the differences in aMPV-pathogenesis between these two species.

Pathology and tissue distribution of turkey coronavirus in experimentally infected chicks and turkey poults.

Twenty 1-day-old specific pathogen free chicks and 20 1-day-old commercially derived turkey poults were inoculated with a Brazilian strain of turkey coronavirus (TCoV) to study the pathogenicity and virus distribution up to 14 days post-inoculation by histopathology, immunohistochemistry, reverse transcriptase polymerase chain reaction and sequencing. At 2-14 dpi, TCoV antigens were detected in the paranasal sinus and lachrymal accessory gland (Harderian gland) of infected chicks and in the ileum, ileocaecal junction and caecum of infected poults. Lymphocytic inflammation was present in these tissues. TCoV was re-isolated from pooled tissue suspensions of nasal concha, Harderian gland and paranasal sinus from chicks, as well as from the ileum, ileocaecal junction and caecum of poults, after three consecutive passages in 28-day-old embryonated turkey eggs. Viral RNA corresponding to the spike gene region (1178-2073 genome position) was amplified from the upper respiratory tract of chickens and from the intestinal tract of poults and phylogenetic analysis confirmed the identity as TCoV. This is the first description of TCoV antigens and mRNA in upper respiratory tissues in experimentally infected chickens.

Infectious bronchitis virus in the chicken Harderian gland and lachrymal fluid: viral load, infectivity, immune cell responses, and effects of viral immunodeficiency.

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.

Comparative histopathology of two serotypes of infectious bronchitis virus (T and n1/88) in laying hens and cockerels.

The comparative and sequential histopathology of different tissues of unvaccinated laying hens and cockerels were studied in chickens exposed to T and N1/88 strain of infectious bronchitis virus (IBV). The Harderian gland and trachea of hens and cockerels in both T- and N1/88-infected groups were damaged to a similar extent. The cecum was unaffected for both strains of IBV in both hens and cockerels. The sequential histopathological changes in hens revealed that IBV multiplies initially in the Harderian gland, then in the tracheal mucosa and simultaneously in the kidney and regions of the oviduct such as the magnum, tubular shell gland, and shell gland pouch. In cockerels, IBV multiplies first in the Harderian gland, then simultaneously in the trachea and kidney. Overall, the severity and persistence of lesions were greater in the kidneys of T-infected hens as compared with N1/88-infected hens. However, pathological changes in the kidney were mild in T- and N1/88-infected cockerels.

Histopathology of two serotypes of infectious bronchitis virus in laying hens vaccinated in the rearing phase.

The comparative histopathology of 2 different strains of infectious bronchitis virus (T and N1/ 88) in vaccinated hens was studied at 110 wk of age. The Harderian gland showed similar histopathology in T- and N1/88-infected hens. The trachea and kidney of challenged vaccinated hens were protected to a moderate extent, but the oviduct was protected to only a small extent. The severity and persistence of lesions were greater in tubular shell gland, shell gland pouch, and kidney of the T-infected hens, whereas, for the magnum, N1/88 had a greater effect.

Pathogenesis of chicken anemia virus: comparison of the oral and the intramuscular routes of infection.

The events during the pathogenesis of chicken anemia virus (CAV) infection following intramuscular (IM) and oral inoculation were further elucidated and compared by sequential clinical, pathologic, and morphometric histopathologic evaluations, and by sequential determination of CAV genome concentrations in different organs. Specific-pathogen-free chickens were inoculated by IM or oral routes with the same dose (2 x 10(6) mean tissue culture infective dose [TCID50]) of CAV isolate 03-4876 at 1 day of age. Weights and hematocrits were obtained at 7, 10, 14, 18, 21, 25, and 28 days postinoculation (DPI). Seven birds from each group were necropsied at 7, 10, 14, and 28 DPI, and samples of thymus, Harderian gland, and cecal tonsils (CT) were obtained for histopathologic examination and CAV genome quantification by real-time polymerase chain reaction. Peak CAV genome concentrations were detected in the thymus at 10 and 14 DPI in the IM and orally infected chickens, respectively. High CAV DNA concentrations were maintained throughout the experimental period until 28 DPI, despite specific seroconversion occurring by 14 DPI in the IM-inoculated chickens. CAV was isolated from both orally and IM-infected chickens 28 DPI. Peak CAV genomes in the thymuses of IM and orally infected chickens coincided with peak lymphocyte depletion in these organs. Lymphocyte repopulation of the thymus occurred by 28 DPI in spite of the presence of the virus in the organs of both infected chicken groups. CAV genomes were detected in the CT, but histopathologic changes were not observed. Compared with the IM route of infection, orally infected chickens did not show apparent signs of illness. Clinical parameters, including reduction of weight gains and hematocrits, and gross and histopathologic changes were delayed and less severe in the orally inoculated chickens. This was concurrent with a delay in accumulation of CAV genomes in the thymus of these chickens.

Comparison of the pathogenicity in rats of rat coronaviruses of different neutralization groups.

BACKGROUND AND PURPOSE: Rat coronaviruses (RCVs) are common natural pathogens of rats that cause clinical illness, necrosis, and inflammation of respiratory, salivary, and lacrimal organs. The aim of the study was to determine whether antigenically different strains of RCV vary in their pathogenic potential in rats. METHODS: Neutralization groups were identified by use of RCV strain-specific antisera. Sprague Dawley rats were inoculated oronasally with RCV-SDA, RCV-BCMM, or RCV-W. Histologic examination, immunohistochemical analysis, and reverse transcriptase-polymerase chain reaction analysis were performed on tissues from infected rats. RESULTS: Clinical illness was not evident in any of the inoculated rats. The RCV-SDA strain caused mild lesions in the exorbital gland of one rat. The RCV-BCMM strain caused severe lesions in the Harderian and parotid glands and mild lesions in the exorbital glands, lungs, and nasal mucosa. The RCV-W strain caused severe lesions in the Harderian, exorbital, and parotid glands and mild lesions in the submandibular glands, lungs, and nasal mucosa. The RNA concentration was highest in the Harderian, parotid, and exorbital glands of RCV-BCMM- and RCV-W-infected rats at postinoculation day 7. CONCLUSIONS: Although RCV-SDA, RCV-BCMM, and RCV-W caused different degrees and patterns of lesions, neutralization groups are not useful for predicting the pathogenic potential of a new RCV isolate.

Investigation into avian pneumovirus persistence in poults and chicks using cyclosporin A immunosuppression.

One-day-old poults or two-week old chicks were infected oculonasally with avian pneumovirus. Cloacal swabs were collected for virus isolation as were selected tissues (Harderian gland, turbinates, trachea, lungs and kidneys) from birds killed at regular intervals up to 33 days post infection (p.i.) for poults, and up to 40 days p. i. for chicks. In an attempt to induce virus re-excretion, the T-cell-suppressor cyclosporin A (CSA) was given for 12 days starting from three weeks p.i. in poults and from four weeks p.i. in chicks. Birds were sampled for virus isolations up to day 12 post CSA treatment. Virus was recovered only up to day nine p.i. in poults, and day five p.i. in chicks during the acute phase of the infection. Despite T-cell suppression, there was no evidence of re-excretion of the virus, and hence no evidence for the persistence of virus in the tissues examined.

The effects of cyclosporin A and cyclophosphamide on the populations of B and T cells and virus in the Harderian gland of chickens vaccinated with the Hitchner B1 strain of Newcastle disease virus.

The cellular response to conjunctival vaccination with the Hitchner B1 strain of Newcastle disease virus was studied in the Harderian gland (HG) by immunohistochemistry. Bu-1+ cells and all subpopulations of T cells, (CD3+, CD4+, CD8+, TCR gamma delta, TCR alpha beta 1, and TCR alpha beta 2) were in the interstitial tissue between the ducts and the acini. Plasma cells with cytoplasmic IgM were more dispersed than the other cells and outlined the acini. Bu-1+ cells and all subpopulations of T cells increased at least three-fold after vaccination when compared to uninfected birds on the basis of the average cell counts in sections taken at 3, 5, 7, 10, 14, and 20 days after vaccination. The most marked increase was in the CD8+ cells which increased six-fold. Virus replicated for 10 days in cyclophosphamide (Cy) treated birds and for 7 days in cyclosporin A (CsA) treated birds compared with 5 days in untreated birds. Cy treatment prevented an antibody response to NDV and reduced Bu-1+ and IgM cells in the HG by 20-fold. Cy treatment resulted in a doubling of the number of T cells in the HG but these T cells may have been transiently disabled because it also caused a poor response of the lymphocytes in whole blood to the T cell mitogen concanavalin A (ConA). CsA reduced the T cell numbers in the HG and whole-blood responses to ConA by about 4-fold but T cell numbers rebounded to normal resting values after vaccination with NDV. The clearance time was prolonged either by T cells being less numerous than normal after CsA or being disabled after Cy. T cells, but not B cells, may therefore be essential for virus clearance. CD8+ cells expanded more than CD4+ cells after the vaccination of untreated and CsA-treated birds indicating that CD8+ cells may be key players in vaccinal immunity to NDV.

Newcastle disease virus vaccines: differences between Line C and Line 15I chickens with respect to virus replication and IgA responses in the gut and Harderian gland.

The Ulster 2C and Hitchner B1 strains of Newcastle disease virus were inoculated into inbred White Leghorn birds of the Reaseheath-C and 15I lines by the oculonasal route. Both viruses replicated in the Harderian gland (HG) and induced virus-specific IgA in the tears and bile. Ulster 2C but not Hitchner B1 replicated in the small intestine and induced virus-specific antibody forming cells (AFC) in the small intestine. Line 15I birds produced 120-fold more virus and 13-fold more IgA-AFC in the small intestine than Line C birds. Line C birds produced 20-fold more virus in the HG and at least three-fold higher titres of lacrimal IgA than Line 15I birds. The level of local virus replication in the HG or small intestine, which varied according to the line of bird, positively predicted the local antibody response in the same organ. When low doses of Ulster 2C were inoculated into Line C birds virus replication was low and irregular in birds older than 18 days whereas low doses of Hitchner B1 replicated in all ages of bird.