Life cycle of chicken bursal secretory dendritic cell (BSDC).

The transmission electron microscopy revealed a dendritic cell in the medulla of the chicken bursal follicle. This dendritic cell has a classical secretory machinery; therefore, it has been named a bursal secretory dendritic cell (BSDC). The corticomedullary epithelial arch (CMEA) encloses lymphoid-like cells, which can proliferate and after entering the medulla, begin to differentiate to immature, then mature BSDC, which discharges glycoprotein (gp). With the exhaustion of gp production, the BSDC rapidly transforms into a macrophage-like cell (Mal), which is an activated endocytic cell of innate immunity. The Mal drifts through the follicle-associated epithelium (FAE)-supporting cells into the FAE, and via FAE, the Mal is eliminated in the bursal lumen. The infectious bursal disease virus (IBDV) infection accelerates the maturation process of BSDC precursors, which results in acute emptying of CMEA and subsequently, numerous immature BSDC(s) emerge. The IBDV infection stops the gp discharge, and the gp appears in the virus-containing Mal. The Movat pentachrome staining recognizes the gp in the extracellular spaces of the medulla and after infection in the Mal. The BSDC is the primary target of the IBDV. During IBDV infection, a large number of suddenly formed Mal actively migrate into the cortex, initiating cytokine storm and recruiting heterophil granulocytes. During embryogenesis, the vimentin-positive, possibly embryonic dendritic cells provide a microenvironment for carbohydrate switch. Around hatching, these embryonic, temporary dendritic cells get the Fc receptor, which bind maternal IgY. The posthatched forms of BSDC(s) gradually replace the embryonic ones and bind their own IgY.

Effect of Intermediate Plus Vaccine and vvIBDV on Bursa Secretory Cells and Their Glycoprotein Production.

There are two types of secretory cells in the chicken bursa of Fabricius (BF): (a) interfollicular epithelial cells (IFE), and (b) bursal secretory dendritic cells (BSDC) in the medulla of bursal follicles. Both cells produce secretory granules, and the cells are highly susceptible to IBDV vaccination and infection. Before and during embryonic follicular bud formation, an electron-dense, scarlet-acid fuchsin positive substance emerges in the bursal lumen, the role of which is unknown. In IFE cells, IBDV infection may induce rapid granular discharge, and in several cells, peculiar granule formation, which suggests that the glycosylation of protein is injured in the Golgi complex. In control birds, the discharged BSDC granules appear in membrane-bound and subsequently solubilized, fine-flocculated forms. The solubilized, fine-flocculated substance is Movat-positive and can be a component of the medullary microenvironment, which prevents the medullary B lymphocytes from nascent apoptosis. Vaccination interferes with the solubilization of the membrane-bound substance, resulting in: (i) aggregation of a secreted substance around the BSDC, and (ii) solid lumps in the depleted medulla. The non-solubilized substance is possibly not "available" for B lymphocytes, resulting in apoptosis and immunosuppression. In IBDV infection, one part of the Movat-positive Mals fuse together to form a medullary, gp-containing "cyst". The other part of Mals migrate into the cortex, recruiting granulocytes and initiating inflammation. During recovery the Movat-positive substance appears as solid, extracellular lumps between the cells of FAE and Mals. Possibly the Mals and Movat-positive extracellular lumps glide into the bursal lumen via FAE to eliminate cell detritus from the medulla.

Glycoprotein Production by Bursal Secretory Dendritic Cells in Normal, Vaccinated, and Infectious Bursal Disease Virus (IBDV)-Infected Chickens.

The aim of this study is to follow the gp production in IBDV-vaccinated and challenged birds. The progress of IBDV infection was monitored using anti-VP2 immunocytochemistry, light and transmission electron microscopy. In the medulla of the bursal follicle, the Movat pentachrome staining discovered an extracellular glycoprotein (gp) produced by bursal secretory dendritic cells (BSDCs). The secretory granules of BSDCs either discharge resulting in extracellular gp or fuse together forming intracellular corpuscles. The double fate of granules suggests a dual function of BSDCs: (a.) For the discharged granules, gp contributes to the medullary microenvironment (ME). (b.) The intracellular corpuscles may be the sign of BSDC transformation to a macrophage-like cell (Mal). Infectious bursal disease virus (IBDV) infection accelerates the BSDC transformation to Mal. The decreased number of BSDCs is feedback for the precursor cells of BSDCs lodging in the cortico-medullary epithelial arches (CMEA), where they proliferate. Opening the CMEA, the precursor cells enter the medulla, and differentiate to immature BSDCs. The virus uptake in the corpuscles prevents the granular discharge resulting in the absence of gp and alteration in ME. In vaccine-take birds, the mitotic rate of BSDC precursor cells cannot restore the precursor pool; therefore, in the case of IBDV challenge, the number of newly formed BSDCs is too low for outbreak of clinical disease. The BSDCs, as a primary target of IBDV, may contribute to the long-lasting immunosuppressive status of IBDV-infected chickens.

The bursal secretory dendritic cell (BSDC) and the enigmatic chB6+ macrophage-like cell (Mal).

The bursal secretory dendritic cell (BSDC) was discovered more than 40 yr ago. It is a highly polarized, granulated cell, locating in the medulla of bursal follicle. The cytoplasmic granules either discharge or fuse together forming large, irregular-shaped, dense bodies. Formation of the dense bodies could be the first sign of BSDC transformation to macrophage-like cell (Mal) which is the result of terminal maturation of BSDC. The BSDC is non-phagocytic, unlike Mal. The discharged substance may be attached to the cell membrane (membrane-bound form) and after detaching, appears as a flocculated substance in the extracellular space of medulla. Movat pentachrome staining shows, that this substance is a glycoprotein (gp), which may be contributed to the microenvironment of the medulla. Medullary lymphocytes are floating in the gp. Precursors of the BSDC locate in the corticomedullary epithelial arches, which operate under the effect of Notch/Serrate signaling. The Notch signaling determines the fate of lymphoblast-like precursor cells and inhibits the appearance of immunoglobulin heavy chain. In the arches, the precursor cells proliferate and entering the medulla differentiate. The dense bodies pack the virus particles, which prevents the granular discharge, resulting in disappearance of extracellular gp, but gp emerges inside the virus containing Mal. In infected birds, the Mal contains either apoptotic cells or virus particles. If vaccination or infectious bursal disease virus (IBDV) infection use up the BSDC precursors, the recovery of follicle is critical.

The morphology and differentiation of stromal cells in the cortex of follicles in the bursa of Fabricius of the chicken.

Mesenchymal reticular cells (MRCs) form a supporting system in the cortex of the bursal follicle. The stellate-shaped MRCs exhibit a low electron density, which is helpful for their identification. A remarkable feature of MRC is the formation of multiple blebs in the nuclear envelope. The large, irregularly shaped blebs-which are perinuclear spaces-may be detached from the nuclear membrane, creating a sac-like granular endoplasmic reticulum (GER). Inside the bleb, membrane-bound bodies originate from cytoplasmic impressions. The cytoplasm contains a few round mitochondria, in which the internal membranes form either ovoid vesicles or the entire internal structure is indistinct. These mitochondria may be associated with the blebs. The classical Golgi complex with cis and trans faces cannot be recognized, but the accumulation of very small vesicles occurs around two or three stacked flat cisterns. The MRC forms a continuous layer along the corticomedullary basal lamina (CMBL), and during cell migration between the cortex and medulla, it may contribute to the temporary closure of the gap in the CMBL. At the outer surface of the cortex, transitory cells between the MRC and fibrocytes of the interfollicular connective tissue are present, and both cells can produce GER by blebbing. This finding suggests that MRCs and fibrocytes may have a common origin. The other stromal cell is the macrophage (Ma), which may fuse together to form multinucleated giant cells. The definition of histological classification of the third type of stromal cell is questionable, but certain morphological features may be referred to as progenitors of MRCs.

Infection of bursal disease virus abrogates the extracellular glycoprotein in the follicular medulla.

In the medulla of bursal follicle, only the secretory dendritic cell (BSDC) is furnished with secretory machinery. The granular discharge of BSDC appears in membrane-bound and solubilized forms. Movat pentachrome staining proves that the solubilized form is a glycoprotein, which fills up the extracellular space of follicular medulla. The glycoprotein contributes to bursal microenvironment and may be attached to the surface of medullary lymphocytes. The secretory granules of BSDC may be fused, resulting in large, irregular dense bodies, which are the first sign of BSDC transformation to macrophage-like cells (Mal). To determine the effect of infectious bursal disease virus (IBDV) infection on the extracellular glycoprotein and BSDC, SPF chickens were experimentally infected with IBDV. On the surface of BSDC, the secretory substance is in high concentration, which may contribute to primary binding of IBDV to BSDC. The early distribution of IBDV infected cells is in consent with that BSDC. The IBDV infected BSDC rapidly transforms to Mal in which the glycoprotein staining appears. In the dense bodies, the packed virus particles inhibit the virus particles preventing the granular discharge, which may represent the first, early phase of virus replication cycle. The absence of extracellular glycoprotein results in alteration in the medullary microenvironment and subsequently B cell apoptosis. On the surface of medullary B cells, the solubilized secretory substance can be in much lower concentration, which results in secondary binding of IBDV to B cells. In secondary, late phase of virus replication cycle, the virus particles are not packed in electron dense substance which results in cytolytic lymphocytes and presence of virus in extracellular space. The Mal emigrates into the cortex, where induces inflammation, recruiting heterophil granulocyte and monocyte.

Avian coronavirus infection induces mannose-binding lectin production in dendritic cell precursors of chicken lymphoid organs.

The aim of this immunocytochemical study was to compare mannose-binding lectin (MBL) production induced by avian coronavirus in the spleen and caecal tonsil (CT). One-day-old specific-pathogen-free (SPF) chickens were experimentally infected with six QX field isolates and the H120 vaccine strain. In the negative control birds, the spleen was MBL negative, while the CT showed scattered MBL-positive cells in close proximity and within the surface epithelium and germinal centre (GC)-like cell clusters. MBL was detectable in the ellipsoid-associated cells (EACs) and cell clusters in the periarterial lymphoid sheath (PALS) by 7 days post infection (dpi). In both organs, the MBL-positive cells occupy antigen-exposed areas, indicating that GC formation depends on resident precursors of dendritic cells. The majority of MBL-positive EACs express the CD83 antigen, providing evidence that coronavirus infection facilitated the maturation of dendritic cell precursors. Surprisingly, co-localisation of MBL and CD83 was not detectable in the CT. In the spleen (associated with circulation), the EACs producing MBL and expressing CD83 are a common precursor of both follicular (FDC) and interdigitating dendritic cells (IDC). In the CT (gut-associated lymphoid tissue, GALT) the precursors of FDC and IDC are MBL-producing cells and CD83-positive cells, respectively. In the CT the two separate precursors of lymphoid dendritic cells provide some 'autonomy' for the GALT.

Effect of IBDV infection on the interfollicular epithelium of chicken bursa of Fabricius.

In the chicken bursa of Fabricius (BF), the interfollicular epithelium (IFE) consists of cylindrical- and cuboidal-shaped cells. Among the cylindrical-shaped epithelial cells, mucus-producing and caveolin-1 (Cav-1)-expressing cells can be distinguished. Occasionally, the cuboidal-shaped cells also express Cav-1, which suggests that they are precursors of both mucus-producing and Cav-1-expressing cells. Very virulent infectious bursal disease virus (IBDV) impedes the differentiation of Cav-1-expressing cells and shifts the differentiation of cuboidal cells towards mucus-producing cells. In control birds exclusively, the IFE surface shows a mucous membrane, but after IBDV infection, the surfaces of both IFE and FAE are also covered by a mucous membrane. After IBDV infection, the cells of FAE also produce mucus, providing evidence for cell transformation. In late postinfection (pi; 28 d pi), the Cav-1 expression returned in the IFE cells, whereas the follicle (the primary lymphoid organ) underwent atrophy. The appearance of the renewed Cav-1-positive cells is similar to that of the normal basal cell, but they randomly locate in different levels of IFE, suggesting the loss of epithelial polarity. Between days 2 and 7 pi, the Cav-1 expression in the endothelial cells of the cortico-medullary capillary web is variable, which may explain the hemorrhage in several infected birds. The IBDV infection stops the Cav-1 expression and subsequently the cholesterol efflux into the bursal lumen. In the infected birds, the high cholesterol level may further worsen the clinical syndrome of IBDV.

Coronavirus infection retards the development of the cortico-medullary capillary network in the bursa of Fabricius of chicken.

Coronavirus infection delays the development of the cortico-medullary (CM) capillary network which results in retarded development of bursal follicles. The smaller size of the medulla in the coronavirus-infected birds may lead to a lower number of B lymphocytes and bursal secretory dendritic cells, which negatively affects the reactivity and efficacy of the immune system. Contrary to the wild-type infectious bronchitis virus (IBV) strain, infection induced by H120 vaccine virus exerts only a moderate influence on caveolin-1 expression of the CM capillary web and on follicular development compared to the untreated controls.

Expression of caveolin-1 in the interfollicular but not the follicle-associated epithelial cells in the bursa of fabricius of chickens.

The surface epithelium of the bursa of Fabricius consists of interfollicular (IFE) and follicle-associated epithelium (FAE). The IFE comprises (i) cylindrical-shaped secretory cells (SC) and (ii) cuboidal basal cells (BCs). The FAE provides histological and two-way functional connections between the bursal lumen and medulla of the follicle. We used a carbon solution and anti-caveolin-1 (Cav-1) to study the endocytic activity of FAE. Carbon particles entered the intercellular space of FAE, but the carbon particles were not internalized by the FAE cells. Cav-1 was not detectable in the FAE cells or the medulla of the bursal follicle. The absence of Cav-1 indicates that no caveolin-mediated endocytosis occurs in the FAE cells, B cells, bursal secretory dendritic cells (BSDC), or reticular epithelial cells. Surprisingly, a significant number of Cav-1 positive cells can be found among the SC, which are designated SC II. Cav-1 negative cell are called SC I, and they produce mucin for lubricating the bursal lumen and duct. Occasionally, BCs also express Cav-1, which suggests that BC is a precursor of a SC. Transmission electron microscopy confirmed the existence of type I and II SC. The SC II are highly polarized and have an extensive trans-Golgi network that is rich in different granules and vesicles. Western blot analysis of bursa lysates revealed a 21-23 kDa compound (caveolin) and Filipin fluorescence histochemistry provided evidence for intracellular cholesterol. High amount of cholesterol in the feces shows the cholesterol efflux from SC II. The presence of Cav-1 and cholesterol in SC II indicates, that the bursa is a complex organ in addition to possessing immunological function contributes to the cholesterol homeostasis in the chickens.

Body temperature and motion: Evaluation of an online monitoring system in pigs challenged with Porcine Reproductive & Respiratory Syndrome Virus.

Highly contagious and emerging diseases cause significant losses in the pig producing industry worldwide. Rapid and exact acquisition of real-time data, like body temperature and animal movement from the production facilities would enable early disease detection and facilitate adequate response. In this study, carried out within the European Union research project RAPIDIA FIELD, we tested an online monitoring system on pigs experimentally infected with the East European subtype 3 Porcine Reproductive & Respiratory Syndrome Virus (PRRSV) strain Lena. We linked data from different body temperature measurement methods and the real-time movement of the pigs. The results showed a negative correlation between body temperature and movement of the animals. The correlation was similar with both body temperature obtaining methods, rectal and thermal sensing microchip, suggesting some advantages of body temperature measurement with transponders compared with invasive and laborious rectal measuring. We also found a significant difference between motion values before and after the challenge with a virulent PRRSV strain. The decrease in motion values was noticeable before any clinical sign was recorded. Based on our results the online monitoring system could represent a practical tool in registering early warning signs of health status alterations, both in experimental and commercial production settings.

Dual secretion locations on type II cells in the avian lung suggest local as well as general roles of surfactant.

Transmission electron microscopy indicates that the avian lung surfactant may be secreted in two directions: a) into air passages of parabronchus, atrium and infundibulum where it forms a trilaminar substance serving the respiratory role and b) to the basolateral surface-intercellular space-of type II pneumocytes, contributing to the innate and adoptive immune responses of lung. Basolateral secretion may be confirmed by the presence of trilaminal substance in the intercellular space of type II pneumocytes. Fusion of surfactant containing vesicles with the lateral plasma membrane may result in membrane fusion of neighboring cells and subsequently formation of multinucleated giant cell. The indistinct and in some places discontinuous basal lamina in the parabronchial atrium and infundibulum permits the hydrophilic surfactant proteins to spread into the interstitium of air-blood capillary region. The hydrophilic surfactant proteins may activate lung interstitial macrophages to migrate into the air passages where they appear as "free avian respiratory macrophages." Therefore, in the interstitium the hydrophilic surfactant proteins are essential soluble components of innate immunity. J. Morphol. 277:1062-1071, 2016. © 2016 Wiley Periodicals, Inc.

Molecular investigations on the prevalence and viral load of enteric viruses in pigs from five European countries.

Enteric viral infections in pigs may cause diarrhea resulting in ill-thrift and substantial economic losses. This study reports the enteric infections with porcine astrovirus type 4 (PAstV4), porcine group A rotavirus (GARV), porcine group C rotavirus (GCRV), porcine circovirus type 2 (PCV2) and porcine kobuvirus (PKoV) in 419 pigs, comprising both healthy and diarrheic animals, from 49 farms in five European countries (Austria, Germany, Hungary, Spain and Sweden). Real-time RT-PCR assays were developed to test fecal samples and to compare the prevalence and viral load in relation to health status, farms of origin and age groups. The results showed that PAstV4 (70.4%) was the dominant virus species, followed by PKoV (56.7%), PCV2 (42.2%), GCRV (3%) and GARV (0.9%). Diarrheic pigs had a higher viral load of PAstV4 in the nursery and growing-finishing groups. Rotaviruses were mainly detected in diarrheic pigs, whereas PCV2 was more often detected in clinically healthy than in diarrheic pigs, suggesting that most PCV2 infections were subclinical. PAstV4, PCV2 and PKoV were considered ubiquitous in the European pig livestock and co-infections among them were frequent, independently of the disease status, in contrast to a low prevalence of classical rotavirus infections.

Naturally occurring parvoviral infection in Hungarian broiler flocks.

The major enteric disease (ED) complex in broiler chickens is runting-stunting syndrome and in turkey broilers is poult enteritis mortality syndrome. Viruses from numerous families have been identified in the intestinal tracts of poultry with ED, such as Astroviridae, Coronaviridae, Reoviridae, Rotaviridae, and Parvoviridae. The objective of the present study was to directly demonstrate the presence of the scarcely known chicken parvovirus (ChPV) and turkey parvovirus (TuPV) in Hungarian flocks experiencing clinical signs of ED. ChPV and TuPV infection were demonstrated in 15 chicken flocks and two turkey flocks, in intestinal samples collected between 2008 and 2010. The histopathological investigation revealed enteritis in the duodenum and jejunum, and atrophy of the lymphoid organs. Indirect immunohistochemistry (IHC) suggested the intestinal epithelium of chickens and turkeys as a potential replication site of the virus, similarly to other parvoviruses, while in case of the turkey samples IHC positivity was also observed in the bursa of Fabricius, liver and pancreas. However, no direct connection could be established between the presence of the pathogen in the above-mentioned tissues and the histopathological changes observed in the investigated flocks. The phylogenetic analysis performed on the partial nucleic acid sequence of the NS1 gene revealed an evident clustering tendency of the ChPV and TuPV strains, but also highlighted the potential reciprocal role of these two species in the epidemiology of these viruses. The role of the ChPV and TuPV in the ED is far from understood, but the results of the present study emphasize the fact that in certain, still not fully elucidated conditions, ChPV and TuPV may participate in the emergence of ED in chicken flocks, as suggested by previous experimental infections.

Comparison of the pathogenicity of QX-like, M41 and 793/B infectious bronchitis strains from different pathological conditions.

Five QX-like infectious bronchitis virus (IBV) strains, isolated from different field outbreaks and two reference IBV strains of known serotypes (M41 and 793/B), were used in the present study to investigate and compare their pathogenicity for 1-day-old specific pathogen free chickens. The ability of the strains to inhibit trachea epithelium ciliary activity, to induce immune response, to replicate and to cause histopathological lesions in designated organs was followed by repeated samplings during a period of 42 days post infection. Clear differences in pathogenicity and in organ distribution of the three serotypes were found. Strain 793/B had the least capacity to invade the investigated organs, while it produced a good humoral response as measured by enzyme-linked immunosorbent assay. The QX-like strains generally replicated to higher titres, although differences were found among the five strains in their pathogenicity and affinity for different organs. The Chinese isolate of QX-like virus caused the most severe lesions and induced the highest antibody titres. Severe kidney damage and dilatation of the oviduct were the prominent lesions that could be related to the QX-like IBV strains, although neither marked virus replication nor histopathological lesions were detected in the oviduct.

Short beak and dwarfism syndrome of mule duck is caused by a distinct lineage of goose parvovirus.

From the early 1970s to the present, numerous cases of short beak and dwarfism syndrome (SBDS) have been reported in mule ducks from France. The animals showed strong growth retardation with smaller beak and tarsus. It was suggested that the syndrome was caused by goose parvovirus on the basis of serological investigation, but the causative agent has not been isolated and the disease has not so far been reproduced by experimental infection. The aim of the present study was to characterize the virus strains isolated from field cases of SBDS, and to reproduce the disease experimentally. Phylogenetic analysis proved that the parvovirus isolates obtained from SBDS of mule duck belonged to a distinct lineage of goose parvovirus-related group of waterfowl parvoviruses. The authors carried out experimental infections of 1-day-old, 2-week-old and 3-week-old mule ducks by the oral route with three different parvovirus strains: strain D17/99 of goose parvovirus from Derzsy's disease, strain FM of Muscovy duck parvovirus from the parvovirus disease of Muscovy ducks, and strain D176/02 isolated from SBDS of mule duck. The symptoms of SBDS of the mule duck could only be reproduced with the mule duck isolate (strain D176/02) following 1-day-old inoculation. Infection with a genetically different strain of goose parvovirus isolated from classical Derzsy's disease (D17/99) or with the Muscovy duck parvovirus strain (FM) did not cause any clinical symptoms or pathological lesions in mule ducks.

Development of a one-step real-time quantitative PCR assay based on primer-probe energy transfer for the detection of porcine reproductive and respiratory syndrome virus.

A one-step real-time RT-PCR method has been developed for the simultaneous detection of both genotypes of porcine reproductive and respiratory syndrome virus (PRRSV). The assay is based on primer-probe energy transfer, and the most important advantage of this is the relative tolerance towards mutations in the target-probe region. The primers and the probe were designed using an alignment of 235 Type 1 (including all subtypes) and Type 2 PRRSV strains. According to the alignment, multiple degenerations were included in the forward and reverse primers to enable the detection of all PRRSV strains deposited in the GenBank. Specificity was tested using 37 different PRRSV strains and eight other swine pathogen viruses. The detection limit was approximately 10 copies of RNA prepared from the Lelystad virus, a European Subtype 3 virus (Belarus strain Soz-8), and an American vaccine virus (Ingelvac MLV). One TCID(50) was the detection limit in the case of the cell cultured Lelystad virus and an American wild type isolate, respectively. The melting point analysis revealed melting point decrease, but no significant sensitivity and signal loss in the presence of numerous (up to five) target-probe mismatches, indicating the capability of tolerating even more mutations. The method was suitable for the detection and quantitation of phylogenetically divergent strains and can serve as a robust, high throughput tool for molecular diagnosis of the PRRSV.