Immunopathogenesis of the Canadian Delmarva (DMV/1639) infectious bronchitis virus (IBV): Impact on the reproductive tract in layers.

IBV infection may lead to reduced egg production and poor egg quality in layer flocks. The DMV/1639 strain was recently identified as one of the most dominant IBV variants isolated from Canadian layer flocks with egg production problems. The current study aimed to investigate the immunopathogenesis of the Canadian DMV/1639 strain in laying chickens. Specific-pathogen-free (SPF) layers were infected at the peak of lay (29 weeks; n = 10) with an uninfected control group (n = 10). Egg production in the infected group dropped to 40% by the fifth day post-infection (dpi). Five birds from the infected and the control groups were euthanized at 5 and 10 dpi. Ovarian regression and shortened oviduct with marked histopathological changes were observed in the infected group at 10 dpi. An increase in the IBV viral load in reproductive tissues was accompanied by a significant recruitment (p < 0.05) of KUL01+ macrophages and CD4+ and CD8+ T cell subsets at 10 dpi. Additionally, anti-IBV antibody response was detected in serum and locally in the reproductive tract washes of the infected group. Overall, our findings contribute to the understanding of the pathogenicity of the Canadian DMV/1639 strain and the subsequent host responses in the reproductive tract of chickens.

Single stranded (ss)RNA-mediated antiviral response against infectious laryngotracheitis virus infection.

BACKGROUND: Single stranded ribonucleic acid (ssRNA) binds to toll-like receptor (TLR)7 leading to recruitment of immune cells and production of pro-inflammatory cytokines, which has been shown in mammals. In chickens, synthetic ssRNA analog, resiquimod, has been shown to elicit antiviral response against infectious bursal disease virus infection. The objective of this study was to determine the innate host responses activated by the pre-hatch in ovo administration of resiquimod against infectious laryngotracheitis virus (ILTV) infection in chickens post-hatch. RESULTS: First, we observed that in ovo treatment of resiquimod at embryo day (ED) 18 increases macrophage recruitment in respiratory and gastrointestinal tissues of chicken day 1 post-hatch in addition to interleukin (IL)-1ß in lungs. Second, we observed that in ovo treatment of resiquimod reduces ILTV cloacal shedding at 7 days post-infection (dpi) when challenged at day 1 post-hatch coinciding with higher macrophage recruitment. In vitro, we found that resiquimod enhances production of nitric oxide (NO) and IL-1ß and not type 1 interferon (IFN) activity in avian macrophages. Although, the antiviral response against ILTV is associated with the enhanced innate immune response, it is not dependent on any of the innate immune mediators observed as has been shown in vitro using avian macrophage. CONCLUSION: This study provides insights into the mechanisms of antiviral response mediated by resiquimod, particularly against ILTV infection in chicken.

Potential mediators of in ovo delivered double stranded (ds) RNA-induced innate response against low pathogenic avian influenza virus infection.

BACKGROUND: Toll like receptor (TLR) 3 is a critically important innate pattern recognizing receptor that senses many viral infections. Although, it has been shown that double stranded (ds) RNA can be used for the stimulation of TLR3 signaling pathway in a number of host-viral infection models, it's effectiveness as an antiviral agent against low pathogenic avian influenza virus (LPAIV) needs further investigation. METHODS: In this study, first, we delivered TLR3 ligand, dsRNA, in ovo at embryo day (ED)18 since in ovo route is routinely used for vaccination against poultry viral and parasitic infections and infected with H4N6 LPAIV 24-h post-treatment. A subset of in ovo dsRNA treated and control groups were observed for the expressions of TLR3 and type I interferon (IFN)s, mRNA expression of interleukin (IL)-1ß and macrophage recruitment coinciding with the time of H4N6 LPAIV infection (24 h post-treatment). Additionally, Day 1 chickens were given dsRNA intra-tracheally along with a control group and a subset of chickens were infected with H4N6 LPAIV 24-h post-treatment whereas the rest of the animals were observed for macrophage and type 1 IFN responses coinciding with the time of viral infection. RESULTS: Our results demonstrate that the pre-hatch treatment of eggs with dsRNA reduces H4N6 replication in lungs. Further studies revealed that in ovo delivery of dsRNA increases TLR3 expression, type I IFN production and number of macrophages in addition to mRNA expression of IL-1ß in lung 24-h post-treatment. The same level of induction of innate response was not evident in the spleen. Moreover, we discovered that dsRNA elicits antiviral response against LPAIV correlating with type I IFN activity in macrophages in vitro. Post-hatch, we found no difference in H4N6 LPAIV genome loads between dsRNA treated and control chickens although we observed higher macrophage recruitment and IFN-ß response coinciding with the time of viral infection. CONCLUSIONS: Our findings imply that the TLR3 ligand, dsRNA has antiviral activity in ovo and in vitro but not in chickens post-hatch and dsRNA-mediated innate host response is characterized by macrophage recruitment and expressions of TLR3 and type 1 IFNs.

Comparative features of infections of two Massachusetts (Mass) infectious bronchitis virus (IBV) variants isolated from Western Canadian layer flocks.

BACKGROUND: Infectious bronchitis virus (IBV) is one of the leading causes of mortality and morbidity in chickens. There are numerous serotypes and variants, which do not confer cross protection resulting in failure of currently used IBV vaccines. Although variant IBV isolates with major genetic differences have been subjected to comparative studies, it is unknown whether minor genetic differences in IBV variants within a serotype are different in terms of pathogenesis and eliciting host responses. Two Massachusetts (Mass) variant IBV isolates recovered from commercial layer flocks in the Western Canadian provinces of Alberta (AB) and Saskatchewan (SK) were compared genetically and evaluated for their pathogenicity, tissue distribution and ability to recruit and replicate in macrophages. RESULTS: Although whole genome sequencing of these two Mass IBV isolates showed low similarity with the M41 vaccinal strain, they had an identical nucleotide sequence at open reading frames (ORFs) 3a, 3b, envelop (E), matrix (M), 5a and 5b. The rest of the ORFs of these 2 IBV isolates showed 99.9% nucleotide similarity. However, upon experimental infection, we found that the IBV isolate originating from AB was different to the one that originated in SK due to higher tracheal lesion scores and lower lung viral replication and lower genome loads in cecal tonsils. Nevertheless, both IBV isolates elicited host responses characterized by significant macrophage recruitment to the respiratory tract and there was evidence that both IBV isolates replicated within tracheal and lung macrophages. CONCLUSIONS: Overall, this study shows that Mass variant IBV isolates, although possessing minor genetic variations, can lead to significant differences in pathogenicity in young chickens. Further studies are required to investigate the pathogenicity of these two Mass variant IBV isolates in laying hens.

Toll-like receptor (TLR)21 signalling-mediated antiviral response against avian influenza virus infection correlates with macrophage recruitment and nitric oxide production.

Cytosine-guanosinedeoxynucleotide (CpG) DNA can be used for the stimulation of the toll-like receptor (TLR)21 signalling pathway in avian species which ultimately leads to up-regulation of gene transcription for pro-inflammatory molecules including nitric oxide and recruitment of innate immune cells. The objective of this study was to determine the antiviral effect of NO, produced in response to in ovo delivery of CpG DNA, against avian influenza virus (AIV) infection. We found that when CpG DNA is delivered at embryo day (ED)18 in ovo and subsequently challenged with H4N6 AIV at ED19 pre-hatch and day 1 post-hatching, CpG DNA reduces H4N6 AIV replication associated with enhanced NO production and macrophage recruitment in lungs. In vitro, we showed that NO originating from macrophages is capable of eliciting an antiviral response against H4N6 AIV infection. This study provides insights into the mechanisms of CpG DNA-mediated antiviral response, particularly against AIV infection in avian species.

Activation of toll-like receptor signaling pathways leading to nitric oxide-mediated antiviral responses.

Toll-like receptors (TLRs), well-characterized pattern-recognizing receptors of the innate arm of the immune system, are vital in detecting pathogen-associated molecular patterns (PAMPs). The TLR-PAMP interaction initiates an intracellular signaling cascade, predominantly culminating in upregulation of antiviral components, including inducible nitric oxide synthase (iNOS). After activation, various TLR pathways can promote iNOS production via the myeloid differentiation primary response-88 (MyD-88) adapter protein. Subsequently, iNOS facilitates production of nitric oxide (NO), a highly reactive and potent antiviral molecule that can inhibit replication of RNA and DNA viruses. Furthermore, NO can diffuse freely across cell membranes and elicit antiviral mechanisms in various ways, including direct and indirect damage to viral genomes. This review emphasizes current knowledge of NO-mediated antiviral responses elicited after activation of TLR signaling pathways.

Comparison of quantitative PCR and digital PCR assays for quantitative detection of infectious bronchitis virus (IBV) genome.

The quantitative polymerase chain reaction (qPCR) technique is an extensively used molecular tool for the detection and quantification of viral genome load. However, since the qPCR assay is a relative quantification method that relies on an external calibration curve it has a lower assay precision and sensitivity. The digital PCR (dPCR) technique is a good alternative to the qPCR assay as it offers highly precise and direct quantification of viral genome load in samples. In this study, performance characteristics such as the quantification range, sensitivity, precision, and specificity of the dPCR technique was compared to qPCR technique for the detection and quantification of IBV genome loads in serial dilutions of IBV positive plasmid DNA, and IBV infected chicken tissue and swab samples. The quantification range of the qPCR assay was wider than that of the dPCR assay, however dPCR had a higher sensitivity compared to qPCR. The precision of quantification of DNA in plasmid samples in terms of repeatability and reproducibility of results was higher when using the dPCR assay compared to qPCR assay. The quantification results of IBV genome load in infected samples by the qPCR and dPCR assays displayed a high correlation. Hence, our findings suggest that dPCR could be used in avian virology research for improved precision and sensitivity in detection and quantification of viral genome loads.

Determination of the virulence status of Clostridium perfringens strains using a chicken intestinal ligated loop model is important for understanding the pathogenesis of necrotic.

Necrotic enteritis (NE) is a poultry intestinal disease caused by virulent strains of the bacterium Clostridium perfringens (C. perfringens). This anaerobic bacterium produces a wide range of enzymes and toxins in the gut which leads to NE development. It is generally accepted by the poultry veterinarians that netB-positive C. perfringens strains are virulent and netB-negative strains do not cause NE. However, NE pathogenesis remains unclear as contradictory results have been reported. The use of experimental in vivo models is a valuable tool to understand the pathogenesis of a disease. In this study, a chicken ligated loop model was used to determine the virulence status of 79 C. perfringens strains from various geographical locations, sources, and genotype profiles. According to our model and based on histologic lesion scoring, 9 C. perfringens strains were classified as commensal, 35 as virulent, and 34 as highly virulent. The virulence of only 1 C. perfringens strain could not be classified as its lesion score was variable (from <10 to >15). In general, NE lesions were more severe in intestinal loops inoculated with netB-positive C. perfringens strains than those inoculated with netB-negative strains. The prevalence of netB among strains classified as commensal, virulent, and highly virulent was 56% (5/9), 54%, (19/35), and 59% (20/34). These results suggest that NetB is not required to cause NE lesions and that other factors are also involved. The classification of the virulence status of C. perfringens strains should not be based solely on the presence or absence of this toxin. Therefore, the use of an in vivo model is essential to distinguish commensal from virulent strains of C. perfringens.

Differential Impact of Massachusetts, Canadian 4/91, and California (Cal) 1737 Genotypes of Infectious Bronchitis Virus Infection on Lymphoid Organs of Chickens.

Infectious bronchitis virus (IBV) induces severe economic losses in chicken farms due to the emergence of new variants leading to vaccine breaks. The studied IBV strains belong to Massachusetts (Mass), Canadian 4/91, and California (Cal) 1737 genotypes that are prevalent globally. This study was designed to compare the impact of these three IBV genotypes on primary and secondary lymphoid organs. For this purpose, one-week-old specific pathogen-free chickens were inoculated with Mass, Canadian 4/91, or Cal 1737 IBV variants, keeping a mock-infected control. We examined the IBV replication in primary and secondary lymphoid organs. The molecular, histopathological, and immunohistochemical examinations revealed significant differences in lesion scores and viral distribution in these immune organs. In addition, we observed B-cell depletion in the bursa of Fabricius and the spleen with a significant elevation of T cells in these organs. Further studies are required to determine the functional consequences of IBV replication in lymphoid organs.

Host Immune Response Modulation in Avian Coronavirus Infection: Tracheal Transcriptome Profiling In Vitro and In Vivo.

Infectious bronchitis virus (IBV) is a highly contagious Gammacoronavirus causing moderate to severe respiratory infection in chickens. Understanding the initial antiviral response in the respiratory mucosa is crucial for controlling viral spread. We aimed to characterize the impact of IBV Delmarva (DMV)/1639 and IBV Massachusetts (Mass) 41 at the primary site of infection, namely, in chicken tracheal epithelial cells (cTECs) in vitro and the trachea in vivo. We hypothesized that some elements of the induced antiviral responses are distinct in both infection models. We inoculated cTECs and infected young specific pathogen-free (SPF) chickens with IBV DMV/1639 or IBV Mass41, along with mock-inoculated controls, and studied the transcriptome using RNA-sequencing (RNA-seq) at 3 and 18 h post-infection (hpi) for cTECs and at 4 and 11 days post-infection (dpi) in the trachea. We showed that IBV DMV/1639 and IBV Mass41 replicate in cTECs in vitro and the trachea in vivo, inducing host mRNA expression profiles that are strain- and time-dependent. We demonstrated the different gene expression patterns between in vitro and in vivo tracheal IBV infection. Ultimately, characterizing host-pathogen interactions with various IBV strains reveals potential mechanisms for inducing and modulating the immune response during IBV infection in the chicken trachea.

Heterologous maternal antibodies derived from infectious bronchitis vaccines prevent the development of lesions associated with false layer syndrome.

Infectious bronchitis virus (IBV) strains of the Delmarva (DMV)/1639 genotype have been causing false layer syndrome (FLS) in the Eastern Canadian layer operations since the end of 2015. FLS is characterized by the development of cystic oviducts in layer pullets infected at an early age. Currently, there are no homologous vaccines for the control of this IBV genotype. Our previous research showed that a heterologous vaccination regimen incorporating Massachusetts (Mass) and Connecticut (Conn) IBV types protects layers against DMV/1639 genotype IBV. The aim of this study was to investigate the role of maternal antibodies conferred by breeders received the same vaccination regimen in the protection against the development of DMV/1639-induced FLS in pullets. Maternal antibody-positive (MA+) and maternal antibody-negative (MA-) female progeny chicks were challenged at 1 day of age and kept under observation for 16 weeks. Oviductal cystic formations were observed in 3 of 14 birds (21.4 %) in the MA- pullets, while the lesions were notably absent in the MA+ pullets. Milder histopathological lesions were observed in the examined tissues of the MA+ pullets. However, the maternal derived immunity failed to demonstrate protection against the damage to the tracheal ciliary activity, viral shedding, and viral tissue distribution. Overall, this study underscores the limitations of maternal derived immunity in preventing certain aspects of viral pathogenesis, emphasizing the need for comprehensive strategies to address different aspects of IBV infection.

Protection of laying chickens against the Canadian DMV/1639 infectious bronchitis virus infection through priming with heterologous live vaccine and boosting with heterologous or homologous inactivated vaccine.

The emergence of the Canadian Delmarva (DMV)/1639 infectious bronchitis virus (IBV) type strains was associated with egg production disorders in Eastern Canadian layer operations. While developing vaccines for novel IBV variants is not typically a reasonable approach, the consideration of an autogenous vaccine becomes more appealing, particularly when the new variant presents significant economic challenges. The current study aimed to compare the efficacies of two vaccination programs that included heterologous live priming by Massachusetts (Mass) and Connecticut (Conn) type vaccines followed by either a commercial inactivated Mass type vaccine or a locally prepared autogenous inactivated DMV/1639 type vaccine against DMV/1639 IBV challenge. The protection parameters evaluated were egg production, viral shedding, dissemination of the virus in tissues, gross and microscopic lesions, and immunological responses. The challenge with the DMV/1639 caused severe consequences in the non-vaccinated laying hens including significant drop in egg production, production of low-quality eggs, serious damage to the reproductive organs, and yolk peritonitis. The two vaccination programs protected the layers from the poor egg-laying performance and the pathology. The vaccination program incorporating the autogenous inactivated DMV/1639 type vaccine was more effective in reducing vial loads in renal and reproductive tissues. This was associated with a higher virus neutralization titer compared to the group that received the commercial inactivated Mass type vaccine. Additionally, the autogenous vaccine boost led to a significant reduction in the viral shedding compared to the non-vaccinated laying hens. However, both vaccination programs induced significant level of protection considering all parameters examined. Overall, the findings from this study underscore the significance of IBV vaccination for protecting laying hens.

Comparative pathogenicity of CA1737/04 and Mass infectious bronchitis virus genotypes in laying chickens.

Infectious bronchitis virus (IBV) is a respiratory virus causing atropism in multiple body systems of chickens. Recently, the California 1737/04 (CA1737/04) IBV strain was identified as one of the circulating IBV variants among poultry operations in North America. Here, the pathogenicity and tissue tropism of CA1737/04 IBV strain in specific-pathogen-free (SPF) hens were characterized in comparison to Massachusetts (Mass) IBV. In 30 weeks-old SPF hens, Mass or CA1737/04 IBV infections were carried out, while the third group was maintained as a control group. Following infection, we evaluated clinical signs, egg production, viral shedding, serology, necropsy examination, and histopathology during a period of 19 days. Also, certain tissue affinity parameters were investigated, which involved the localization of viral antigens and the detection of viral RNA copies in designated tissues. Our findings indicate that infection with CA1737/04 or Mass IBV strain could induce significant clinical signs, reduced egg production, and anti-IBV antibodies locally in oviduct wash and systemically in serum. Both IBV strains showed detectable levels of viral RNA copies and induced pathology in respiratory, renal, enteric, and reproductive tissues. However, the CA1737/04 IBV strain had higher pathogenicity, higher tissue tropism, and higher replication in the kidney, large intestine, and different segments of the oviduct compared to the Mass IBV strain. Both IBV strains shed viral genome from the cloacal route, however, the Mass IBV infected hens shed higher IBV genome loads via the oropharyngeal route compared to CA1737/04 IBV-infected hens. Overall, the current findings could contribute to a better understanding of CA1737/04 IBV pathogenicity in laying hens.

Detection and Phylogenetic Analysis of Orf Virus and Muskox Rhadinovirus 1 from Muskoxen (Ovibos moschatus) in the Canadian Arctic.

Orf virus (genus Parapoxvirus) has been associated with gross skin lesions on muskoxen (Ovibos moschatus) from Victoria Island, Nunavut, Canada, where muskox populations are experiencing population declines. Orf virus causes painful proliferative and necrotizing dermatitis upon viral replication and shedding, which may lead to animal morbidity or mortality through secondary infections and starvation. Herpesvirus, known to cause gross lesions on skin and mucosa during active viral replication, has also been documented in muskoxen but to date has not been associated with clinical disease. Our objective was to characterize the variation of orf virus and herpesvirus in wild muskoxen of the Canadian Arctic. Tissue samples including gross skin lesions from the nose, lips, and/or legs were opportunistically collected from muskoxen on Victoria Island, Nunavut and Northwest Territories, and mainland Nunavut, Canada, from 2015 to 2017. Sampled muskoxen varied in age, sex, location, hunt type, and body condition. Tissues from 60 muskoxen were tested for genetic evidence of orf virus and herpesvirus infection using PCR targeting key viral genes. Tissues from 38 muskoxen, including 15 with gross lesions, were also examined for histological evidence of orf virus and herpesvirus infection. Eleven muskoxen (10 from Victoria Island and one from mainland Nunavut) with gross lesions had microscopic lesions consistent with orf virus infection. Muskox rhadinovirus 1, a gammaherpesvirus endemic to muskoxen, was detected in 33 (55%) muskoxen including 17 with gross lesions. In all tissues examined, there was no histological evidence of herpesvirus-specific disease. Sequencing and characterization of amplified PCR products using phylogenetic analysis indicated that a strain of orf virus, which appears to be unique, is likely to be endemic in muskoxen from Victoria Island and mainland Nunavut. Many of the muskoxen are also subclinically infected with a known muskox-endemic strain of herpesvirus.

Efficacy of Two Vaccination Strategies against Infectious Bronchitis in Laying Hens.

Vaccination remains the leading control method against infectious bronchitis (IB) in poultry despite the frequently observed IB outbreaks in vaccinated flocks. Here, two vaccination regimes were evaluated against challenge with the Massachusetts (Mass) infectious bronchitis virus (IBV) strain that was linked to egg production defects in Western Canada. One vaccination strategy included live attenuated IB vaccines only, and the other used both inactivated and live attenuated IB vaccines. The two immunization programs involved priming with a monovalent live attenuated IB vaccine (Mass serotype) at day-old, followed by intervals of bivalent live attenuated IB vaccines containing the Mass and Connecticut (Conn) serotypes given to the pullets at 2-, 5-, 9-, and 14-week-old. Inactivated IB vaccine (Mass serotype) was administrated to only one group of the vaccinated birds at 14-week-old. At the peak of lay, the hens were challenged with the Mass IBV isolate (15AB-01) via the oculo-nasal route. The efficacy of the vaccines was assessed following the challenge by observing clinical signs, egg production, egg quality parameters, seroconversion, and systemic T-cell subsets (CD4+ and CD8+ cells). Moreover, the viral genome loads in the oropharyngeal (OP) and cloacal (CL) swabs were quantified at predetermined time points. At 14 days post-infection (dpi), all the hens were euthanized, and different tissues were collected for genome load quantification and histopathological examination. Post-challenge, both vaccination regimes showed protection against clinical signs and exhibited significantly higher albumen parameters, higher anti-IBV serum antibodies, and significantly lower levels of IBV genome loads in OP swabs (at 3 and 7 dpi) and trachea and cecal tonsils compared to the mock-vaccinated challenged group. However, only the birds that received live attenuated plus inactivated IB vaccines had significantly lower IBV genome loads in CL swabs at 7 dpi, as well as decreased histopathological lesion scores and IBV genome loads in magnum compared to the mock-vaccinated challenged group, suggesting a slightly better performance for using live attenuated and inactivated IB vaccines in combination. Overall, the present findings show no significant difference in protection between the two vaccination regimes against the Mass IBV challenge in laying hens.

An Investigation of the Antiviral Potential of Phytocompounds against Avian Infectious Bronchitis Virus through Template-Based Molecular Docking and Molecular Dynamics Simulation Analysis.

Vaccination is widely used to control Infectious Bronchitis in poultry; however, the limited cross-protection and safety issues associated with these vaccines can lead to vaccination failures. Keeping these limitations in mind, the current study explored the antiviral potential of phytocompounds against the Infectious Bronchitis virus using in silico approaches. A total of 1300 phytocompounds derived from fourteen botanicals were screened for their potential ability to inhibit the main protease, papain-like protease or RNA-dependent RNA-polymerase of the virus. The study identified Methyl Rosmarinate, Cianidanol, Royleanone, and 6,7-Dehydroroyleanone as dual-target inhibitors against any two of the key proteins. At the same time, 7-alpha-Acetoxyroyleanone from Rosmarinus officinalis was found to be a multi-target protein inhibitor against all three proteins. The potential multi-target inhibitor was subjected to molecular dynamics simulations to assess the stability of the protein-ligand complexes along with the corresponding reference ligands. The findings specified stable interactions of 7-alpha-Acetoxyroyleanone with the protein targets. The results based on the in silico study indicate that the phytocompounds can potentially inhibit the essential proteins of the Infectious Bronchitis virus; however, in vitro and in vivo studies are required for validation. Nevertheless, this study is a significant step in exploring the use of botanicals in feed to control Infectious Bronchitis infections in poultry.

The impact of the experimental route of challenge on the host responses and pathogenesis of the Canadian Delmarva (DMV/1639) infectious bronchitis virus infection in laying chickens.

Infectious bronchitis virus (IBV) infection can be associated with respiratory, renal, and/or reproductive diseases in chickens. Under natural conditions, conjunctiva, mucosa of upper respiratory tract, and cloaca are the main routes of IBV entry. Experimentally, the study of IBV infection involved various routes of inoculation. This study investigated the impact of adding the trachea as a potential route of viral entry to the oculo-nasal infection on the host responses, pathogenicity, and tissue tropism of the Canadian IBV Delmarva (DMV/1639) strain in laying chickens. Specific-pathogen-free laying chickens were divided into three experimental groups: control group (Con group), oculo-nasal challenged group (ON group), and oculo-nasal/intratracheal challenged group (ON/IT group); all groups were observed for 12 days post-infection (dpi). The clinical signs and reduction in egg production in the ON/IT group started slightly earlier compared to the ON group. At 12 dpi, the gross lesions in the ON/IT group were confined to the ovary, while the ON group showed regressed ovary and atrophied oviduct. Only the ON group showed significantly higher microscopic lesion scores in the lung, kidney, magnum, and uterus compared to the control group at 12 dpi. The oviduct tissues of the ON group showed a significant increase in B cells infiltration compared to ON/IT and control groups. The viral shedding (detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR)), tissue tropism (detected either by qRT-PCR or immunohistochemistry (IHC)), T/natural killer cells infiltration in reproductive tract (detected by IHC), and antibody-mediated immune responses (measured by enzyme-linked immunosorbent assay) showed similar patterns in the ON and ON/IT groups.

The tumor microenvironment generated by Marek's disease virus suppresses interferon-gamma-producing gamma delta T cells.

The tumor microenvironment (TME) is generated by the cross-talk among tumor cells, immune system cells, and stromal cells. The TME generated by Marek's disease virus (MDV) is suggested to display an immunosuppressive milieu due to immune inhibitory molecules and cytokines which are possibly induced by MDV-transformed cells and regulatory T cells. Both anti-tumor and pro-tumor gamma delta (γδ) T cells are reported in human cancer. Although anti-tumor like and pro-tumor like γδ T cells are found in MDV-infected chickens at the later phase of infection, how the TME affects circulating and tissue-resident γδ T cells has not been investigated. Here, we demonstrated that the supernatant of the cultured splenocytes derived from MDV-challenegd chickens inhibited interferon (IFN)-γ production and CD25 expression by T cell receptor (TCR)γδ-stimulated tissue-resident γδ T cells, but the supernatant of the cultured MDV-transformed cell line did not affect γδ T cell activation. TCRγδ-stimulated circulating γδ T cells were influenced neither by the supernatant of the cultured splenocytes derived from MDV-challenegd chickens nor by the supernatant of the cultured MDV-transformed cell line. Taken together, activation and IFN-γ production by tissue-resident γδ T cells can be inhibited in the TME generated by MDV while tumor attracted circulating γδ T cells may not be influenced in activation and IFN-γ production by the TME generated by MDV.

Replication of infectious bronchitis virus (IBV) Delmarva (DMV)/1639 variant in primary and secondary lymphoid organs leads to immunosuppression in chickens.

Infectious bronchitis virus (IBV) that primarily causes respiratory infection in chickens, disseminate to multiple body systems leading to pathology, results in economic losses to poultry industry. IBV replicates in the bursa of Fabricius (BF), Harderian gland (HG), cecal tonsils (CT), and spleen. The objective of this study was to investigate the immunosuppressive effect of IBV Delmarva (DMV/1639) variant in chickens. Specific pathogen free chickens were infected with the IBV DMV/1639 variant while maintaining an age-matched uninfected control group. At predetermined time points, subsets of the infected and control chickens were observed for changes in body weights and pathological changes. The histopathological lesions were observed in the CT and BF, with minimal lesions in the thymus and spleen. The mRNA expression of pro-inflammatory mediators suggested immunomodulation by IBV, favoring viral replication. Further studies are warranted to observe the functional impact of the IBV DMV/1639 variant's replication in immune organs.

In Ovo Vaccination with Recombinant Herpes Virus of the Turkey-Laryngotracheitis Vaccine Adjuvanted with CpG-Oligonucleotide Provides Protection against a Viral Challenge in Broiler Chickens.

Infectious laryngotracheitis (ILT) is an economically important disease in chickens. We previously showed that an in ovo adjuvantation of recombinant herpesvirus of the turkey-Laryngotracheitis (rHVT-LT) vaccine with CpG-oligonucleotides (ODN) can boost vaccine-induced responses in one-day-old broiler chickens. Here, we evaluated the protective efficacy of in ovo administered rHVT-LT + CpG-ODN vaccination against a wild-type ILT virus (ILTV) challenge at 28 days of age and assessed splenic immune gene expression as well as cellular responses. A chicken-embryo-origin (CEO)-ILT vaccine administered in water at 14 days of age was also used as a comparative control for the protection assessment. The results showed that the rHVT-LT + CpG-ODN or the CEO vaccinations provided significant protection against the ILTV challenge and that the level of protection induced by both the vaccines was statistically similar. The protected birds had a significantly upregulated expression of interferon (IFN)γ or interleukin (IL)-12 cytokine genes. Furthermore, the chickens vaccinated with the rHVT-LT + CpG-ODN or CEO vaccine had a significantly higher frequency of γδ T cells and activated CD4+ or CD8+ T cells, compared to the unvaccinated-ILTV challenge control. Collectively, our findings suggest that CpG-ODN can be used as an effective adjuvant for rHVT-LT in ovo vaccination to induce protective immunity against ILT in broiler chickens.