Chicken Immune Cell Assay to Model Adaptive Immune Responses In Vitro

Knowledge about the modes of action of immunomodulating compounds such as pathogens, drugs, or feed additives, e.g., probiotics, gained through controlled but animal-related in vitro systems using primary cultured peripheral blood mononuclear cells (PBMCs) will allow the development of targeted nutrition strategies. Moreover, it could contribute to the prevention of infectious diseases and the usage of antimicrobials, and further promote the health of the animals. However, to our knowledge, a protocol for the isolation of PBMCs with reduced thrombocyte count from chicken blood and subsequent cell culture over several days to assess the effects of immunomodulating compounds is not available. Therefore, we established an optimized protocol for blood sampling and immune cell isolation, culture, and phenotyping for chicken PBMCs. For blood sampling commercial Na–citrate tubes revealed the highest count of vital cells compared to commercial Li–heparin (p < 0.01) and K3EDTA (p < 0.05) tubes. Using combined dextran and ficoll density gradient separation, the thrombocyte count was significantly reduced (p < 0.01) compared to slow-speed centrifugation with subsequent ficoll. For cell culture, the supplementation of RPMI-1640 medium with 10% chicken serum resulted in the lowest relative cell count of thrombocytes compared to fetal calf serum (FCS) (p < 0.05). To validate the ability of the cell culture system to respond to stimuli, concanavalin A (conA) was used as a positive control. The optimized protocol allows the isolation and cultivation of vital PBMCs with reduced thrombocyte count from chicken blood for subsequent investigation of the modes of action of immunomodulating compounds.

Mechanical Examination of Chicken Vessel with Custom-Built Experimental Equipment

Cardiovascular disease has been decimating humanity for decades. In vivo examination of blood vessels is of great help in the development of numerical models and simulations that can help physicians significantly improve sufferers’ quality of life. For such models, the different mechanical characteristics of the vessels are the input data. Several such mechanical properties of the vessels, such as modulus of elasticity and tensile strength, are determined by a tensile test. In the course of our research, an experimental device was developed and tested which is suitable for biaxial tensile tests of blood vessels, which we present through the examination of chicken blood vessels.

A Rapid and Simple Assay Correlates In Vitro NetB Activity with Clostridium perfringens Pathogenicity in Chickens

Necrotic enteritis is an important enteric disease in poultry, caused by NetB-producing Clostridium (C.) perfringens strains. As no straight-forward method to assess the NetB activity of C. perfringens was available, we aimed to develop an easy, high-throughput method to measure the NetB activity produced by C. perfringens. First, the appearance of C. perfringens on different avian blood agar plates was assessed. Based on the size of the haemolysis surrounding the C. perfringens colonies, NetB-positive strains could phenotypically be discriminated from NetB-negative strains on both chicken and duck blood agar. Additionally, strains producing the consensus NetB protein induced more pronounced haemolysis on chicken blood agar as compared to the weak outer haemolysis induced by A168T NetB-variant-producing C. perfringens strains. Next, a 96-well plate-based haemolysis assay to screen NetB activity in the C. perfringens culture supernatants was developed. Using this assay, a positive correlation between the in vitro NetB activity and virulence of the C. perfringens strains was shown. The developed activity assay allows us to screen novel C. perfringens isolates for their in vitro NetB activity, which could give valuable information on their disease-inducing potential, or identify molecules and (bacterial) metabolites that affect NetB expression and activity.

Effect of blood source on vector competence of Culex pipiens biotypes for Usutu virus

Background Infectious blood meal experiments have been frequently performed with different virus-vector combinations to assess the transmission potential of arthropod-borne (arbo)viruses. A wide variety of host blood sources have been used to deliver arboviruses to their arthropod vectors in laboratory studies. The type of blood used during vector competence experiments does not always reflect the blood from the viremic vertebrate hosts in the field, but little is known about the effect of blood source on the experimental outcome of vector competence studies. Here we investigated the effect of avian versus human blood on the infection and transmission rates of the zoonotic Usutu virus (USUV) in its primary mosquito vector Culex pipiens. Methods Cx. pipiens biotypes (pipiens and molestus) were orally infected with USUV through infectious blood meals containing either chicken or human whole blood. The USUV infection and transmission rates were determined by checking mosquito bodies and saliva for USUV presence after 14 days of incubation at 28 °C. In addition, viral titers were determined for USUV-positive mosquito bodies and saliva. Results Human and chicken blood lead to similar USUV transmission rates for Cx. pipiens biotype pipiens (18% and 15%, respectively), while human blood moderately but not significantly increased the transmission rate (30%) compared to chicken blood (17%) for biotype molestus. USUV infection rates with human blood were consistently higher in both Cx. pipiens biotypes compared to chicken blood. In virus-positive mosquitoes, USUV body and saliva titers did not differ between mosquitoes taking either human or chicken blood. Importantly, biotype molestus had much lower USUV saliva titers compared to biotype pipiens, regardless of which blood was offered. Conclusions Infection of mosquitoes with human blood led to higher USUV infection rates as compared to chicken blood. However, the blood source had no effect on the vector competence for USUV. Interestingly, biotype molestus is less likely to transmit USUV compared to biotype pipiens due to very low virus titers in the saliva.

A practical, low-cost, short-term storage method for genomic DNA

The aim of this study was to assess the DNA preservation capability of cellulose paper towel and blotting paper as low-cost alternatives to commercial DNA preservation products. Chicken blood was applied as DNA source to each paper towel, blotting paper, FTA® cards and DNA/RNA Shield™. All samples were stored at room temperature for 130 days. DNA extraction from dried blood spots was performed after various time periods using Tris–EDTA and NaOH protocols. PCR activity and the mean amount of DNA isolated from paper towels were reliable. The results of this study demonstrated that cellulose-based blotting paper and especially paper towel had considerable DNA binding and preservation capacity for at least 130 days at room temperature without DNA degradation.

ANALISIS FAKTOR TITIK KRITIS DAN UJI MALACHITE GREEN UNTUK MENENTUKAN STATUS HALAL AYAM POTONG DI TPA KECAMATAN MENES

This study aims to analyze the critical point factor and malachite green test to determine the halal status of broiler chickens in chicken slaughterhouses (TPA). Samples were taken from 4 landfills in Menes District. The research was conducted in 3 stages, namely first filling the halal slaughtering quisoner according to the LPPOM MUI standard (2011), with results of 80% in accordance with the halal chicken slaughtering technique. The second stage is the Malachite Green test, which aims to prove whether the process of slaughtering broiler chickens is perfect, seen from the removal of chicken blood which must also be perfect, the data obtained is that blood removal is carried out completely from all samples (negative carcass). The last stage is post-slaughter handling by testing Eacherichia coli microbial contamination. The average value of microbial contamination is 2.6 x 104 cfu / gr with purple colonies on brilliance media, and the amount of e.coli contamination of broiler chicken meat exceeds the maximum limit of Eacherichia Coli microbial contamination (BMCM) of fresh chicken meat is less than 1 x 101 cfu / gr.

The role of corticosterone in the regulation of the cellular composition of chicken blood during the stress reaction

The influence of hen layer density on the variability of the number of red blood cells, heterophiles and lymphocytes in the blood, the secretory activity of adrenal glands, estimated by the level of corti-costerone and cortisol, as well as the presence of interrelations between hormones and blood cells by calculating complex indices, were studied. Chickens, as the research object, were kept in cages, under conditions of standard layer density and increased by 1.5 and 2.0 times. We found that chickens adapt to an increase in layer density by one and a half times, pro-vided that egg production decreases to 33.33%; two times exceed of the regulatory requirements for laying does not correspond to the adaptive abilities of birds. Depending on the level of layer density excess (stress factor) in chicken blood, the concentration of corticosterone and cortisol increases, determining a decrease in the number of lymphocytes and an in-crease in heterophiles against the background of the preservation of red blood cells, reflecting the “energy price” of adaptation. Corticosterone af-fects the relationship of red blood cells with lymphocytes and heterophiles, determining the variability of the values of the indices reflecting the ratio of red blood cells and lymphocytes (ISEL), red blood cells and hetero-philes (ISEG), red blood cells, lymphocytes and corticosterone (ISELC), red blood cells, heterophiles and corticosterone (ISEGC) and the integral index of red blood cells-heterophiles-lymphocytes and corticosterone (IIEGLC).