Avian Macrophage Responses to Virulent and Avirulent Clostridium perfringens

The present study evaluated the avian macrophage responses against Clostridium perfringens that varied in their ability to cause necrotic enteritis in chickens. Strains CP5 (avirulent-netB+), CP1 (virulent-netB+), and CP26 (highly virulent-netB+tpeL+) were used to evaluate their effect on macrophages (MQ-NCSU cells) and primary splenic and cecal tonsil mononuclear cells. The bacilli (whole cells) or their secretory products from all three strains induced a significant increase in the macrophage transcription of Toll-like receptor (TLR)21, TLR2, interleukin (IL)-1β, inducible nitric oxide synthase (iNOS), and CD80 genes as well as their nitric oxide (NO) production and major histocompatibility complex (MHC)-II surface expression compared to an unstimulated control. The CP1 and CP26-induced expression of interferon (IFN)γ, IL-6, CD40 genes, MHC-II upregulation, and NO production was significantly higher than that of CP5 and control groups. Furthermore, splenocytes and cecal tonsillocytes stimulated with bacilli or secretory products from all the strains showed a significant increase in the frequency of macrophages, their surface expression of MHC-II and NO production, while CP26-induced responses were significantly higher for the rest of the groups. In summary, macrophage interaction with C. perfringens can lead to cellular activation and, the ability of this pathogen to induce macrophage responses may depend on its level of virulence.

A Case of Newcastle disease from an outbreak in a commercial broiler farm in Mymensingh city, Bangladesh

Background Newcastle disease (ND) is a contagious and often deadly viral disease caused by the Newcastle disease virus (NDV), affecting a wide variety of domestic and wild birds. The outbreak of this fatal disease is one of the greatest constraints to the expansion of poultry farms, resulting in significant financial losses. Here we report the clinical and pathological features of a ND case from an outbreak in a commercial broiler farm. Materials and Methods A broiler farm with a population of 850 birds aged 27 days reported the death of 100 chickens within 4 days of the onset of the disease in 2019. For investigation, one dead chicken was brought to the department of pathology, Bangladesh Agricultural University, Mymensingh. The case history was recorded, and an autopsy was performed. Portion of the samples were kept in 10% neutral buffered formalin for histopathological study. Results The morbidity and mortality rates were reported to be 17.65% and 11.47%, respectively. Recorded clinical history were depression, off-feed, huddling, gasping, ruffled feathers, greenish diarrhea, soiled vent and the birds were unvaccinated. On external examination, the birds appeared dehydrated, dyspneic and had nasal exudates, ruffled feathers, and soiled vents. Autopsy exhibited prominent gross lesions in the trachea, lungs, proventriculus, gizzard, intestine, cecal tonsil, liver, spleen and cloacal bursa. Grossly, tracheal hemorrhage, severe congestion in the lungs, pin point hemorrhages on the tip of the proventriculur glands, hemorrhage in the cecal tonsil, button-like ulceration in the intestine and mottled spleen were suggestive of ND. Histopathologically, severe enteritis, necrotic mass in the cecal tonsil and proventriculus, lymphoid depletion in the spleen supported the infection of NDV. The clinicopathological findings of the ND outbreak in broiler farm confirmed that it was velogenic viscerotropic in nature. Conclusion ND in commercial flocks remains a threat to the poultry industry in Bangladesh. Implementation of strict biosecurity, husbandry practice and effective vaccination are required to prevent diseases and improve economic stability.

Morphohistology and Biometric Characteristics of Cecal Tonsils of Sonali Chicken at Post-Hatching Ages

Knowledge of basic structures is prerequisite for acquiring an in-depth idea about the physiology and immunology of the lymphoid system. The study evaluates the age related histomorphometry of cecal tonsil of Sonali chicken at different postnatal stages in Bangladesh as literatures regarding this are very scarce. The investigation was carried out on 25 healthy Sonali chickens representing different stage of postnatal life: days 1, 14, 28, 42, and 56 (n=5). After ethically sacrifice (cervical subluxation method), cecal tonsil was collected and subjected for both gross and histological studies. Haematoxylin and Eosin stain was done for microscopic study. Morphologically, cecal tonsils were located bilaterally at the junction of small and large intestine. It had tubular structure and yellowish white in color. All gross parameters (weight, length, and width) found to be increased significantly (P<0.05) throughout the whole study period. Weight was measured 0.022±0.001 g at day 1 and noticed 0.181±0.016 g at the end of study tenure. The microscopic observations revealed that at day 28 encapsulated lymphatic nodules was present along with the diffuse lymphocytes at the lamina propria and submucosa layer, which was absent at the previous study groups. At day 1, only small infiltration of lymphocytes was identified and at day 14, lymphocytes were aggregating to form lymphatic nodules. After that, age related development was noticed in histological features. The findings would be a milestone to give an idea about the gut health and immune status of Sonali chicken and provide a basis for further immunization research.

SOCS3 Promotes ALV-J Virus Replication via Inhibiting JAK2/STAT3 Phosphorylation During Infection

Avian leukosis virus subgroup J (ALV-J) is an oncogenic retrovirus that causes immunosuppression and neoplastic diseases in poultry. Cytokine signal-transduction inhibitor molecule 3 (SOCS3) is an important negative regulator of the JAK2/STAT3 signaling pathway and plays certain roles in ALV-J infection. It is of significance to confirm the roles of SOCS3 in ALV-J infection and study how this gene affects ALV-J infection. In this study, we assessed the expression of the SOCS3 gene in vivo and in vitro, and investigated the roles of SOCS3 in ALV-J infection using overexpressed or interfered assays with the SOCS3 in DF-1 cells. The results showed that the SOCS3 expression of ALV-J infected chickens was different from uninfected chickens in the spleen, thymus and cecal tonsil. Further, SOCS3 is mainly expressed in the nucleus as determined by immunofluorescence assay. Overexpression of SOCS3 in DF-1 cells promoted the replication of ALV-J virus, and the expression of interferons (IFNα and INFβ), inflammatory factors (IL-6 and TNFα) along with interferon-stimulating genes (CH25H, MX1, OASL, and ZAP). Conversely, interference of SOCS3 showed the opposite results. We also observed that SOCS3 promoted ALV-J virus replication by inhibiting JAK2/STAT3 phosphorylation. In conclusion, SOCS3 promotes ALV-J replication via inhibiting the phosphorylation of the JAK2/STAT3 signaling pathway. These results would advance further understanding of the persistent infection and the viral immune evasion of the ALV-J virus.

Tolerogenic Immunoregulation towards Salmonella Enteritidis Contribute to Colonization Persistence in Young Chicks

Long-term survival and the persistence of the bacteria in the host suggest either host unresponsiveness or induction of immunological tolerant response towards the pathogen. The role of host immunological response to persistent colonization of Salmonella Enteritidis (SE) in chicken remains poorly understood. In the current study, we performed a cecal tonsil transcriptome analysis in a two-week-old SE persistent infection model to comprehensively examine the dynamic of host immunological responses in the chicken gastrointestinal tract. Our results revealed an overall host tolerogenic adaptive immune regulation occurrence in one of the major gut-associated lymphoid tissue, cecal tonsil, during SE infection. Specifically, we observed consistent down-regulation of metallothionein 4 at all four post-infection time points (3,7,14 & 21dpi) which suggested potential pathogen-associated manipulation of the host zinc regulation as well as possible immune modulatory effect. Furthermore, delayed activation in the B cells receptor signaling pathway and failure to sustain its active state during the lag phase of infection was further supported by an insignificant production of both intestinal and circulatory antibody levels. Tag of war for IL-2 regulation between effector T cells and regulatory T cells appear to have the consequential outcome on up-regulation in Transducer of ERBB2 also known as TOB pathway, a negative regulator of T cell proliferation. In conclusion, current work highlighted the overall host tolerogenic immune response that promoted persistent colonization of SE in young layer chicks.

Efficacy of a nanoparticle vaccine administered in-ovo against Salmonella in broilers

Salmonella is a zoonotic pathogen that persists in poultry. Salmonella vaccines that can be delivered in-ovo can be cost-effective and can decrease Salmonella load in poultry. This study evaluates the efficacy of a Salmonella chitosan-nanoparticle (CNP) vaccine, administered in-ovo, in broilers. CNP vaccine was synthesized with Salmonella Enteritidis (SE) outer-membrane-proteins (OMPs) and flagellin proteins. At embryonic-d18, one-hundred-thirty-six eggs were injected with 200μl PBS or 1000μg CNP into the amniotic cavity. At d1-of-age, 132 chicks were allocated in 6 pens/treatment with 11 chicks/pen. At d7, birds were orally challenged with 1×109 CFU/bird SE. At d1, 8h-post-challenge, d14, and d21, serum anti-SE-OMPs IgY were analyzed. At d14 and d21, cloacal swabs and bile anti-SE-OMPs IgA, CD4+/CD8+-T-cell ratios, and ceca SE loads were analyzed. At d21, cecal tonsil IL-1β, IL-10, and iNOS mRNA were analyzed. Body-weight-gain (BWG) and feed-conversion-ratio (FCR) were recorded weekly. Data were analyzed by Student’s t-test at P<0.05. There were no significant differences in BWG or FCR between vaccinated birds compared to control. At d1, CNP-vaccinated birds had 5.62% greater levels (P<0.05) of anti-SE-OMPs IgY, compared to control. At 8h-post-challenge, CNP-vaccinated birds had 6.39% greater levels (P<0.05) of anti-SE-OMPs IgY, compared to control. At 2wk-post-challenge, CNP-vaccinated birds had 7.34% lower levels (P<0.05) of anti-SE-OMPs IgY, compared to control. At 1wk-post-challenge, CNP-vaccinated birds had 15.30% greater levels (P<0.05) of bile anti-SE-OMPs IgA, compared to control. At d14 and d21, CNP-vaccinated birds had 0.62 and 0.85 Log10 CFU/g, decreased SE ceca load (P<0.05), respectively, compared to control. There were no significant differences in CD4+/CD8+-T-cell ratios between vaccinated birds compared to control. There were no significant differences in IL-1β, IL-10, iNOS mRNA between vaccinated birds compared to control. Findings demonstrate that the in-ovo administration of CNP vaccine can induce an antigen-specific immune response against SE and can decrease SE cecal load in broilers.

Probiotic Lactobacilli Limit Avian Influenza Virus Subtype H9N2 Replication in Chicken Cecal Tonsil Mononuclear Cells

Low pathogenic avian influenza virus (LPAIV) H9N2 poses significant threat to animal and human health. The growing interest in beneficial effects of probiotic bacteria on host immune system has led to research efforts studying their interaction with cells of host immune system. However, the role of lactobacilli in inducing antiviral responses in lymphoid tissue cells requires further investigation. The objective of the present study was to examine the antiviral and immunostimulatory effects of lactobacilli bacteria on chicken cecal tonsils (CT) cells against H9N2 LPAIV. CT mononuclear cells were stimulated with probiotic Lactobacillus spp mixture either alone or in combination with a Toll-like receptor (TLR)21 ligand, CpG oligodeoxynucleotides (CpG). Pre-treatment of CT cells with probiotic lactobacilli, alone or in combination with CpG, significantly reduced H9N2 LPAIV replication. Furthermore, lactobacilli alone elicited cytokine expression, including IL-2, IFN-γ, IL-1β, IL-6, and IL-12, and IL-10, while when combined with CpG, a significantly higher expression of (interferon-stimulated gene (viperin)), IL-12, IL-6, CXCLi2, and IL-1β was observed. However, none of these treatments induced significant changes in nitric oxide production by CT cells. In conclusion, probiotic lactobacilli demonstrated a modulatory effect on CT cells, and this correlated with enhanced antiviral immunity and reduced H9N2 LPAIV viral replication.

Morphological and Immunohistochemical Examination of Lymphoproliferative Lesions Caused by Marek’s Disease Virus in Breeder Chickens

Marek’s disease is widely controlled by vaccination programs; however, chickens are not totally protected, especially immediately after the vaccination when a strong challenge could interfere with the effectiveness of vaccination in the absence of proper biosecurity practice. This case report describes the occurrence of Marek’s disease (MD) observed in a breeder chicken flock reared southeast of Sicily. MD outbreak occurred from 32 to 47 weeks with an increase in weekly mortality rate (+0.4–0.6%). Overall, mortality rate related to Marek’s disease was about 6% at the end of the cycle. Carcasses of chickens found during the occurrence of disease underwent necropsy, and tissues were collected to confirm the infection. Gizzard, cecal tonsil, intestine, spleen and tumor mass were collected and analyzed from a carcass of one hen, 32 weeks old and apparently asymptomatic. Multiplex real-time PCR performed on spleen tissues detected the presence of MD virus pathogenic strain. Macroscopic and microscopic evaluation of the rest of the samples confirmed the neoplastic disease. Moreover, the immunophenotype of the tumor cells was identified as CD3 positive by immunohistochemical (IHC) staining. The vaccinated flock had become rapidly infected with the MD virus, which proves that the challenge of the MD virus was too strong in the rearing house at the beginning of the cycle, causing the outbreak.