Baicalin attenuated Mycoplasma gallisepticum-induced immune impairment in chicken bursa of fabricius through modulation of autophagy and inhibited inflammation and apoptosis.

BACKGROUND: Mycoplasma gallisepticum (MG) is the primary etiologic agent of chronic respiratory disease in poultry. However, the mechanism underlying MG-induced immune dysregulation in chicken is still elusive. Baicalin shows excellent anti-bacterial, anti-inflammatory, anti-carcinogenic and anti-viral properties. In the present study, the preventive effects of baicalin against immune impairment in chicken bursa of fabricius (BF) were studied in an MG infection model. RESULTS: Histopathological examination showed increased inflammatory cell infiltrations and fragmented nuclei in the model group. Ultrastructural analysis revealed the phenomenon of apoptosis in bursal cells, along with the deformation of mitochondrial membrane and swollen mitochondria in the model group. However, these abnormal morphological changes were partially alleviated by baicalin. Meanwhile, baicalin treatment attenuated the level of proinflammatory cytokines, and suppressed nuclear factor-kappa B expression at both protein and mRNA level. Terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling assay showed extensive apoptosis in BF in the model group. The mRNA and protein expression levels of apoptosis-related genes were upregulated in BF, while baicalin treatment significantly alleviated apoptosis in BF. In addition, alterations in mRNA and protein expression levels of autophagy-related genes and mitochondrial dynamics proteins were significantly alleviated by baicalin. Moreover, baicalin treatment significantly attenuated MG-induced decrease in CD8+ cells and reduced bacterial load in chicken BF compared to the model group. CONCLUSIONS: These results suggested that baicalin could effectively inhibit MG-induced immune impairment and alleviate inflammatory responses and apoptosis in chicken BF. © 2020 Society of Chemical Industry.

Salmonella infection may alter the expression of toll like receptor 4 and immune related cells in chicken bursa of Fabricius.

Toll like receptor 4 (TLR4), eosinophils and mast cells play significant role in host immunity during several pathogenic infections. However in vivo tissue expression of TLR4 and distribution pattern of eosinophils and mast cells in chicken bursa of Fabricius (BF) during Salmonella enterica serovar Typhimurium (STm) infection is poorly studied. Therefore, herein, following immunostaining, we found localization of TLR4 in follicular cortex and medulla and its expression was statistical increased after 36 h and 72 h of STm stimulation. Chromotrope 2R staining revealed that eosinophils were mostly distributed in follicular cortex, inter-follicular spaces and in or around blood vessels and their number in BF were statistical increased after 72 h of STm stimulation. The presence of eosinophils was confirmed using immunostaining with anti-rabbit eosinophil cationic protein antibody. Toluidine blue stained mast cells were mostly distributed in connective tissues between inter-follicular spaces while some were also present in follicular cortex of BF. However, STm stimulation illustrated non-significant effect on the number of mast cells or their de-granulation, instead their number were gradually decreased in BF with advancement in age of chickens. Hence, this study provided novel information about in vivo tissue distribution of TLR4, eosinophils and mast cells in BF during STm infection.

Influence of the Gut Microbiota Composition on Campylobacter jejuni Colonization in Chickens.

The Campylobacter jejuni-host interaction may be affected by the host's gut microbiota through competitive exclusion, metabolites, or modification of the immune response. To understand this interaction, C. jejuni colonization and local immune responses were compared in chickens with different gut microbiota compositions. Birds were treated with an antibiotic cocktail (AT) (experiments 1 and 2) or raised under germfree (GF) conditions (experiment 3). At 18 days posthatch (dph), they were orally inoculated either with 104 CFU of C. jejuni or with diluent. Cecal as well as systemic C. jejuni colonization, T- and B-cell numbers in the gut, and gut-associated tissue were compared between the different groups. Significantly higher numbers of CFU of C. jejuni were detected in the cecal contents of AT and GF birds, with higher colonization rates in spleen, liver, and ileum, than in birds with a conventional gut microbiota (P < 0.05). Significant upregulation of T and B lymphocyte numbers was detected in cecum, cecal tonsils, and bursa of Fabricius of AT or GF birds after C. jejuni inoculation compared to the respective controls (P < 0.05). This difference was less clear in birds with a conventional gut microbiota. Histopathological gut lesions were observed only in C. jejuni-inoculated AT and GF birds but not in microbiota-colonized C. jejuni-inoculated hatchmates. These results demonstrate that the gut microbiota may contribute to the control of C. jejuni colonization and prevent lesion development. Further studies are needed to identify key players of the gut microbiota and the mechanisms behind their protective role.

Immune complexes of E. coli antigens and maternal IgG in the bursa of Fabricius.

The bursa of Fabricius of the chicken is known to be both a primary lymphoid organ and a secondary lymphoid tissue. Bursal follicles are equipped with antigen-trapping follicle-associated epithelium. However, bioactive antigens such as protein and bacteria have not been detected in the bursal parenchyma. By immunoperoxidase staining with a polyspecific antibody (Ab) against Escherichia coli, we detected aggregated E. coli antigens in the medulla of bursal follicles after hatching. The distribution of aggregated E. coli antigens is restricted to the medulla of bursal follicles. The antigens are not found in the spleen or the parenchyma of the caecal tonsil. The bursa is thus a trapping site for E. coli antigens from the external environment. Furthermore, two-color immunostaining clarified that these antigens form immune complexes with maternal IgG (MIgG) and are retained by reticular cells. Additionally, immune complexes in the bursa were shown to induce the rapid development of serum IgM Ab for indigenous E. coli. Our results suggest that immune complexes of MIgG and environmental antigens in the medulla of bursal follicles exert positive effects on B-cell differentiation in the bursa in situ.

A nonimmunosuppressive helper virus allows high efficiency induction of B cell lymphomas by reticuloendotheliosis virus strain T.

We have documented the effect of two nondefective helper viruses, reticuloendotheliosis virus A (REV-A) and chick syncytial virus (CSV) infection on bursal tissue. REV-A infection results in bursal atrophy, destroying both its structural and functional integrity. In contrast, the bursae in CSV-infected chicks, while reduced slightly in size, appear both structurally and functionally normal. REV-A-induced bursal atrophy is not a result of viral replication in the B-lymphocyte as (a) both viruses are capable of inducing, with equal efficiency, the formation of preneoplastic lesions containing proliferating B lymphocytes and (b) it appears that equivalent amounts of viral antigen are expressed in the bursae of chicks infected with either virus. We have examined the phenotype of tumors induced by the replication-defective virus REV-T when replicated by the two different helper viruses, REV-A and CSV. In REV-T(REV-A)-infected chicks, the majority of tumors that develop are negative for IgM expression. In contrast, the majority of tumors induced by REV-T(CSV) infection are IgM+. This finding is confirmed by recovery of IgM- cell lines from REV-T(REV-A)-infected chicks and IgM+ cell lines from REV-T(CSV)-infected chicks. In addition, repopulation studies show that bursal-derived cells that are IgM+ serve as target cells for REV-T(CSV)-induced lymphomas. This study demonstrates, therefore, that REV-T can induce IgM+, B cell lymphomas with high efficiency. We conclude that infections by the helper viruses, REV-A and CSV, differ dramatically in their effects on the composition of the population of cells that serve as targets for REV-T-induced neoplasia.

Quantification of FITC-labelled probiotic Lactobacillus salivarius DSPV 001P during gastrointestinal transit in broilers.

The knowledge related to the fate of probiotics in the complex environment of the intestinal microbiota in broilers is just beginning to be elucidated; however, it is not yet well understood. A good method to investigate the mechanisms by which probiotics mediate their effects is to mark probiotic bacteria and trace them. The aim of this research was to develop a new method to estimate in vivo fluorescein isothiocyanate (FITC)-labelled Lactobacillus salivarius DSPV 001P counts during passage through the gastrointestinal tract (GIT) of broilers. Forty-five, 1 d old Cobb broilers were used in this trial. Programmed necropsies were performed 30 min, 6 h, and 12 h after the administration of the probiotic bacterium, and samples of liver, crop, duodenum, caecum, and bursa of fabricius were collected. To determine the spatial and temporal transit of L. salivarius DSPV 001P in broilers, the number of bacteria as well as its respective fluorescent signal produced by FITC were measured. In order to observe the relationship between the variables, a logistic regression analysis was applied. The amount of fluorescence could be used as an indicator of fluorescent probiotic bacteria in the crop and duodenum 30 min after probiotic bacterium supplementation. In addition, the fluorescent signal could be used to estimate bacterial counts in caecum 6 and 12 h after L. salivarius DSPV 001P administration. To the best of our knowledge, this research is the first in vivo trial to employ the bacterial FITC-labelling technique in order to enumerate probiotic bacteria during gastrointestinal transit in broilers.

Fermented rapeseed meal is effective in controlling Salmonella enterica serovar Typhimurium infection and improving growth performance in broiler chicks.

The aim of present experiment was to assess the effects of fermented rapeseed meal (FRSM) on Salmonella enterica serovar Typhimurium (S. Typhimurium) colonization and growth performance in broiler chicks. Two hundred forty day-old male Cobb 500 broiler chicks were divided into six experimental treatments with four replicates and 10 birds per each. The treatments were including two positive and negative controls which birds received a basal corn-soybean diet as well as four others which birds received the diets that rapeseed meal (RSM) or FRSM was replaced with soybean meal at 50 and 100% levels. All chicks except the negative control birds were challenged orally with 105 CFU of S. Typhimurium at 3days of age. Results showed that birds were fed FRSM had significantly greater lactic acid bacteria populations and lesser S. Typhimurium colonization in ileal and cecal sections compared to others (P<0.05). The less percentage of liver and bursa of fabricius was belonged to negative control group. At 10day, feeding chicks with diet containing FRSM, but not RSM, significantly decreased the organ invasion by S. Typhimurium (P<0.05). Heterophil to lymphocyte ratio was significantly lesser in chicks were fed FRSM compared to those fed RSM or positive control (P<0.05). Birds were fed FRSM had significantly higher weight gain and better feed conversion ratio compared to those birds were fed RSM (P<0.05). The findings of present experiment concerning positive effects of feeding FRSM on reducing S. Typhimurium and improving growth performance show that this processed protein source can be considered as a nutritional effective strategy to control Salmonella contamination in broiler chicks.

Novel Pathways Revealed in Bursa of Fabricius Transcriptome in Response to Extraintestinal Pathogenic Escherichia coli (ExPEC) Infection.

Extraintestinal pathogenic Escherichia coli (ExPEC) has major negative impacts on human and animal health. Recent research suggests food-borne links between human and animal ExPEC diseases with particular concern for poultry contaminated with avian pathogenic E. coli (APEC), the avian ExPEC. APEC is also a very important animal pathogen, causing colibacillosis, one of the world's most widespread bacterial diseases of poultry. Previous studies showed marked atrophy and lymphocytes depletion in the bursa during APEC infection. Thus, a more comprehensive understanding of the avian bursa response to APEC infection will facilitate genetic selection for disease resistance. Four-week-old commercial male broiler chickens were infected with APEC O1 or given saline as a control. Bursas were collected at 1 and 5 days post-infection (dpi). Based on lesion scores of liver, pericardium and air sacs, infected birds were classified as having mild or severe pathology, representing resistant and susceptible phenotypes, respectively. Twenty-two individual bursa RNA libraries were sequenced, each yielding an average of 27 million single-end, 100-bp reads. There were 2469 novel genes in the total of 16,603 detected. Large numbers of significantly differentially expressed (DE) genes were detected when comparing susceptible and resistant birds at 5 dpi, susceptible and non-infected birds at 5 dpi, and susceptible birds at 5 dpi and 1 dpi. The DE genes were associated with signal transduction, the immune response, cell growth and cell death pathways. These data provide considerable insight into potential mechanisms of resistance to ExPEC infection, thus paving the way to develop strategies for ExPEC prevention and treatment, as well as enhancing innate resistance by genetic selection in animals.

A rapid quantitative method for detecting infectious bursal disease virus using polystyrene latex microspheres.

A monoclonal antibody (mAb) to infectious bursal disease virus (IBDV) was bound to polystyrene latex microspheres. The microspheres agglutinated with extracts of bursae and sera from chickens infected with all strains or isolates of IBDV tested. Agglutination appeared within a 10-min reaction time. The assay could detect a 10(3.7) to 10(4.5) mean embryo infective dose (EID50) of the virus in 0.01 ml and the titer of the assay was 10- to 40-times higher than that of the agar gel precipitin test.

B cell and macrophage response in chicks after oral administration of Salmonella typhimurium strains.

Despite the fact that, in a number of countries, vaccination programmes are extensively used to control Salmonella infection in poultry, information on the immune mechanisms, especially the cellular response, is still needed. The aim of the study was to characterise the B cell and macrophage response in caecum (IgA+, IgM+, IgG+ cells, macrophages), bursa of Fabricius (IgM+ cells, macrophages), and spleen (IgM+ cells) of chicks after oral administration of a non-attenuated Salmonella (S.) typhimurium wild-type strain (infection) or an attenuated commercial live S. typhimurium vaccine strain (immunisation) to day-old chicks as compared to non-treated control birds using immunohistochemistry and image analysis. In caecum, higher counts of IgM-secreting cells were detected in infected animals compared with the controls from day 5 until day 12 of age. In contrast, in treated groups, IgA-secreting cells were found in higher numbers only between day 8 and 12 of age. Infected birds showed a higher number of IgA+ cells in spleen and bursa of Fabricius compared to the controls. In the bursa of Fabricius of immunised and infected birds, a depletion of strongly stained IgM+ cells and macrophages was established between day 5 and 9 indicating a possibly special and independent role of this organ during the immunological reaction against Salmonella organisms. The results suggest that IgM- and IgA-secreting cells are of importance in the caecal immune response of chickens against Salmonella strains. Immunised chickens always showed a weaker immune reaction compared to infected animals. Present findings regarding the B cell reaction within avian caeca prove a participation of both humoral and cellular immunity in defence against Salmonella strains. Immunohistochemical examination of the cellular response (B cells and macrophages) in relevant organs of chickens may be an important tool to evaluate the immunogenic characteristics of potential Salmonella live vaccine candidates.

Extracutaneous viral inclusions in psittacine beak and feather disease.

Thirty-five birds that died with naturally acquired psittacine beak and feather disease (PBFD) were necropsied to identify extracutaneous viral inclusions. Inclusions were found in various tissue sections from 34 of 35 birds. By immunoperoxidase staining, intranuclear and intracytoplasmic inclusion bodies were shown to contain PBFD viral antigen. Inclusion-bearing lesions were widely disseminated but often closely associated with the alimentary tract. Lesions within the palate, esophagus, crop, intestine, bursa of Fabricius, and liver probably serve as sources for viral shedding into the feces.

The pathogenesis of velogenic Newcastle disease virus infection of chickens of different ages and different levels of immunity.

Chickens of 7 weeks or 20 weeks of age were divided into three groups according to their antibody status (high, low, absent) and were infected with a velogenic viscerotropic Newcastle disease virus. To follow patterns of viral replication, birds were necropsied at regular intervals up to 22 days and organs were sampled from each bird. In non-immune birds, virus could be isolated from all organs examined. In birds with antibody, virus was most frequently isolated from the proventriculus, cecal tonsil, bursa, and brain. However, because no one organ could be recommended for all situations, all four should be sampled for field diagnosis. In immune birds, although clinical signs were either mild or absent, widespread virus replication occurred up to 19 days post-challenge.

Detection of infectious bursal disease viruses using in situ hybridization and non-radioactive probes.

The in situ hybridization assay was developed for the detection of infectious bursal disease virus (IBDV) infections in chickens. Bursal tissue samples were harvested 4 days following infection with the ST-C, MD, E, IN, or SAL IBDV strain. The cDNA clones STC-243, located on genome segment A, and STC-119, located on genome segment B, were used to prepare non-radioactive probes. Probes were labeled with digoxigenin and detected the homologous ST-C virus and also heterologous viruses in bursal tissue sections. No positive cells were observed in tissue sections from uninfected control chickens.

Molecular detection of infectious bursal disease virus by polymerase chain reaction.

The polymerase chain reaction (PCR) technique was applied to the detection of infectious bursal disease virus (IBDV). Reverse transcription followed by the PCR was used to amplify a portion of IBDV genome. A set of primers that specify a 150-base-pair segment of IBDV genome was chosen from an Australian strain of IBDV. Standard challenge strain and variant strains A, D, E, G, and GLS-5 of IBDV serotype 1 and OH strain of serotype 2 from infected bursae were subjected to reverse transcription, followed by 30 cycles of PCR. A single band of the PCR product (DNA) of the expected size from each strain of IBDV was visible on polyacrylamide gels stained with ethidium bromide. Using the same primers, no PCR product was detected from genomic nucleic acids of turkey hemorrhagic enteritis virus, infectious bronchitis virus, reovirus, Salmonella enteritidis, Escherichia coli, and uninfected bursae. The PCR could be efficiently performed on serially diluted IBDV RNA and could detect 2 femtograms of IBDV RNA. The identity of the PCR products was confirmed by direct sequencing. The PCR is a specific and sensitive method for the detection of IBDV.

Effect of inv mutations on Salmonella virulence and colonization in 1-day-old White Leghorn chicks.

Invasion of Salmonella into the cells of the intestinal epithelium is an important step in the infection process. This initial invasion is followed by colonization of other organs throughout the body. In an attempt to better understand this process, we moved defined mutations in several genes of the inv locus into Salmonella typhimurium UK-1 and two strains of Salmonella enteritidis. These mutant strains were evaluated for their oral and intraperitoneal virulence as determined by 50% lethal dose in 1-day-old white leghorn chicks. These inv mutants were also studied for their ability to colonize orally infected chicks. The invA, invB, and invC mutations all caused a reduction in oral virulence and colonization by UK-1 and the S. enteritidis strains. Mutation of the invH gene had little or no effect on oral virulence or colonization. None of the inv genes tested had any effect on virulence of these Salmonella strains when administered intraperitoneally.

Infectious bursal disease viral infections. II. The relationship of age, complement levels, virus-neutralizing antibody, clotting, and lesions.

Experimental infection with infectious bursal disease virus (IBDV) reduced the complement (C) titer in 8-week-old chickens on days 3, 5, and 7 postinfection. Since the C titer was much lower in normal 2-week-old chickens than in normal 8-week-old chickens it could not be determined whether there was a reduction in titer during the infection process. Virus-neutralizing antibody rose rapidly following infection in both 2- and 8-week-old chickens. Hyper-immune serum given during infection in an attempt to produce immune complexes did not increase disease severity in either 2- or 6-week-old susceptible chickens.

Immune-complex involvement in the pathogenesis of infectious bursal disease virus in chickens.

Frozen kidney sections from chickens inoculated with infectious bursal disease virus (IBDV) were stained with fluorescein-conjugated rabbit anti-chicken gamma-globulin. Fluorescence was observed in the renal glomeruli of infected chickens, indicating that gamma-globulins, probably in the form of immune complexes, had lodged in the glomeruli of IBDV-infected chickens. This suggests that immune complexes may play an important role in the pathogenesis of IBDV infections in chickens.

An infectious bursal disease virus outbreak in 14- and 15-week-old chickens.

Infectious bursal disease virus (IBDV) observed in a flock of 14- and 15-week-old chickens was typical of the acute symptomatic IBDV infections more common in younger birds. High flock morbidity was indicated by a marked decrease in feed consumption, although deaths were not excessive. At necropsy, affected birds had small hemorrhages in thigh muscles, creamy-yellow-colored bursae of Fabricius with prominent longitudinal striations, and swollen mottled kidneys. Histopathologic examination revealed bursal lesions typical of IBDV infection. One of six sera from necropsied birds was positive for antibody to IBDV in the agar-gel precipitin (AGP) test, and one week later all 35 samples tested were positive. Bursae were homogenized and found to contain IBDV as evidenced by precipitation, with antibody to IBDV, in the AGP test.

Infectious bursal disease viral infections. I. Complement and virus-neutralizing antibody response following infection of susceptible chickens.

Complement (C) titers were decreased at 3 days postinfection, and virus-neutralizing (VN) antibody was detectable at 3 or 4 days postinfection in chickens with infectious bursal disease virus (IBDV). Clotting times were prolonged in all groups tested during the acute phase of the disease. Mortality appeared to be associated with the severity of decrease in C titer. The results suggest that the mortality and many of the clinical signs seen with infectious bursal disease are associated with: 1) a depletion in circulating levels of hemolytic C from the formation of immune complexes at sites of viral replication; and/or 2) a depletion in some clotting factor which results in hemorrhage.

Growth of infectious bursal disease virus with plaque formation in chick embryo fibroblast cell culture.

The WA69 isolant of infectious bursal disease virus (IBDV) induced cytopathic effects and plaque formation in chick embryo fibroblast (CEF) cultures after serial passages in embryonated eggs and then in CEF cultures. The plaque-forming agent was cloned (designated WA69 clone) and identified as IBDV on the basis of serologic response in inoculated birds and its antigenic relationship to other known IBDV isolants. The WA69 clone replicated rapidly in CEF cultures, reaching peak titers at 48 hours postinoculation, and the virus caused only minimal histologic lesions of the bursa when inoculated into 3-week-old chicks from a specific-pathogen-free flock. The growth of IBDV in CEF cultures with plaque formation may provide a simple in vitro system for virological and serological studies of IBDV.