Identification and cloning of two immunogenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO) of C. perfringens

Clostridium-related poultry diseases such as necrotic enteritis (NE) and gangrenous dermatitis (GD) cause substantial economic losses on a global scale. Two antigenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO), were identified by reaction with immune sera from commercial meat-type chickens with clinical outbreak of Clostridium infections. In addition to the genes encoding EF-Tu and PFO, C. perfringens alpha-toxin and necrotic enteritis B-like (NetB) toxin were also expressed in Escherichia coli and their corresponding recombinant proteins were purified. Using the four recombinant proteins as target antigens in ELISA immunoassays, high serum antibody titers were observed not only in chickens with clinical signs of Clostridium infections, but also in apparently healthy animals from the same disease-endemic farm. By contrast, no antibodies against any of the proteins were present in the serum of a specific pathogen-free bird. In ELISA using recombinant proteins of C. perfringens, the levels of anti-bacterial protein antibodies were also higher in chickens which were experimentally induced to show NE clinical signs after co-infection with C. perfringens and Eimeria maxima compared with uninfected controls. These results show that two antigenic C. perfringens proteins, EF-Tu and PFO can be useful detection antigens for C. perfringens-afflicted infections in commercial poultry.     

Development and characterization of mouse monoclonal antibodies reactive with chicken CD80

This study was carried out to develop and characterize mouse monoclonal antibodies (mAbs) against chicken CD80 (chCD80). A recombinant plasmid containing a chCD80/horse IgG4 fusion gene was constructed and expressed in CHO cells to produce recombinant chCD80/IgG4 protein. Chicken CD80 was purified from the chCD80/IgG4 fusion protein following enterokinase digestion, and used to immunize BALB/c mice, resulting in 158 hybridomas that produced mAbs against chCD80. Three mAbs with high binding specificity for recombinant chCD80/IgG4-transfected CHO cells were identified by flow cytometry, and one of these (#112) was selected for further characterization. Immunoprecipitation of CD80/IgG4-CHO cell extract, or lipopolysaccharide (LPS)-treated monocytes identified 35.0 kDa proteins. Immunohistochemical analysis revealed chCD80-expressing cells exclusively in the bursal follicles at the outer portion of the cortex, and throughout the red pulp and the outer boundary of the white pulp in the spleen. By immunofluorescence microscopy, chCD80 was observed on intestinal dendritic cells. LPS treatment of bursa or spleen monocytes for 24 or 48 h increased chCD80 expression. Finally, addition of chCD80 mAb to Con A-stimulated spleen cells inhibited the expression of major histocompatibility complex class II antigens and IL-2-driven proliferation of lymphoblast cells. In summary, these chCD80 mAbs will serve as valuable immunological reagents for basic and applied poultry immunology research.     

Development and characterization of mouse monoclonal antibodies reactive with chicken interleukin-2 receptor αlpha chain (CD25)

This study was carried out to develop and characterize mouse monoclonal antibodies (mAbs) against chicken CD25 (chCD25), the alpha chain of the interleukin-2 (IL-2) receptor. A recombinant chimeric chCD25/IgG4 fusion protein was expressed in Chinese hamster ovary (CHO) cells and isolated from spent cell culture medium by protein G affinity chromatography. Purified chCD25 protein was used to immunize mice, from which 54 stable hybridomas secreting chCD25 mAbs were produced. Two mAbs, chCD25-32 and chCD25-54, with high binding affinity for chCD25-expressing CHO cells were selected for further characterization. By flow cytometry, both mAbs detected cells in the spleen, bursa of Fabricius, intestinal duodenum, and immunostained established chicken T cell, B cell, and macrophage cell lines. Both mAbs reacted with a 55 kDa protein on Western blots of lysates from concanavalin A (Con A)-stimulated spleen mononuclear cells. Intraperitoneal injection of chickens with bacterial lipopolysaccharide increased the percentage of chCD25(+) spleen cells by approximately 4-fold compared with untreated animals. In vitro stimulation of spleen cells with Con A increased the percentage of chCD25(+) cells by up to 50-fold compared with cells treated with medium alone. Finally, the chCD25-32 mAb suppressed IL-2-driven spleen cell proliferation and reduced IL-2-induced nitric oxide production. These mAbs may be useful for future investigation of chicken regulatory T cells.     

May-Hegglin anomaly in a dog

An 8-year-old female spayed Pug dog was presented for evaluation of cutaneous lesions occurring secondary to immunosuppressive treatment of presumed immune-mediated thrombocytopenia. Abnormal hematologic findings included persistent thrombocytopenia, macrothrombocytes, and variably shaped, often fusiform, blue cytoplasmic inclusions in neutrophils. May-Hegglin anomaly (MHA) was suspected based on the morphologic appearance of platelets and neutrophils. Examination of cells by transmission electron microscopy revealed normal platelet ultrastructure; neutrophil inclusions had features similar to those reported for inclusions in human MHA. Neutrophil function was within normal limits based on flow cytometric analysis. Thrombelastography indicated a prolonged clotting time (r), and PlateletMapping showed a lack of response to 2 μM ADP compared with a moderate response in the control dog. Immunocytochemical staining of blood smears using 2 commercially available antibodies against MYH9 protein (nonmuscle myosin heavy chain II) yielded negative results. However, genomic DNA sequencing analysis of the dog's MYH9 gene identified a single point mutation, resulting in substitution of lysine for glutamine at the 1841 amino acid position; this mutation is identical to one identified in people with MHA. To our knowledge, this is the first report of an MYH9 mutation in the dog. MHA-associated macrothrombocytopenia may be mistaken for immune-mediated thrombocytopenia.     

Preparation, characteristics, and stability of glutathione-loaded nanoparticles

The aim of this study was to investigate the characteristics and oxidative stability of chitosan-glutathione conjugate (CS-GSH) and CS-GSH nanoparticles (CS-GSH NPs) to explore the potentials of these nanoparticle systems for GSH delivery. CS-GSH was synthesized using a radical polymerization method, and CS-GSH NP was prepared by ionic gelation of CS-GSH with sodium tripolyphosphate (TPP). The sizes of CS-GSH NPs significantly increased with increasing CS-GSH concentration and CS-GSH/TPP ratio. The entrapment efficiency (EE) significantly increased with increasing CS-GSH concentration and significantly decreased with increasing CS-GSH/TPP ratio. The immobilized GSH could be protected against oxidation compared to free GSH. The thiol content in the nanoencapsulated GSH was more effectively maintained than those in free GSH and CS-GSH, regardless of the presence of oxidative stress-inducing agents. These results suggest that CS-GSH NP can be used to enhance the oxidative stability of GSH.     

Expression patterns of influenza virus receptors in the respiratory tracts of four species of poultry

The primary determinant of influenza virus infectivity is the type of linkage between sialic acid and oligosaccharides on the host cells. Hemagglutinin of avian influenza viruses preferentially binds to sialic acids linked to galactose by an α-2,3 linkage whereas hemagglutinin of human influenza viruses binds to sialic acids with an α-2,6 linkage. The distribution patterns of influenza receptors in the avian respiratory tracts are of particular interest because these are important for initial viral attachment, replication, and transmission to other species. In this study, we examined the distribution patterns of influenza receptors in the respiratory tract of chickens, ducks, pheasants, and quails because these species have been known to act as intermediate hosts in interspecies transmission. Lectin histochemistry was performed to detect receptor-bearing cells. Cell-specific distribution of the receptors was determined and expression densities were compared. We observed species-, site-, and cell-specific variations in receptor expression. In general, receptor expression was the highest in quails and lowest in ducks. Pheasants and quails had abundant expression of both types of receptors throughout the respiratory tract. These results indicate that pheasants and quails may play important roles as intermediate hosts for the generation of influenza viruses with pandemic potential.     

Polymerase chain reaction-based restriction fragment length polymorphism pattern of porcine reproductive and respiratory syndrome virus directly from lung tissues without virus isolation in Korea.

Polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) analysis was developed for directly typing porcine reproductive and respiratory syndrome virus (PRRSV) from lung specimens without virus isolation. Twenty nine lung specimens collected from postweaning pigs were isolated for PRRSV. When the PCR products from the 29 lung specimens were digested by the restriction enzymes MluI, HincII, SacII and HaeIII, the RFLP patterns from the 29 lung specimens matched with those from the corresponding PRRSV isolates from each pig. The results suggest that the PCR-based RFLP analysis method may be useful to distinguish PRRSV isolates directly from lung specimens without virus isolation.     

Effects of dietary lysine supplementation in cats with enzootic upper respiratory disease

To determine the effectiveness of dietary lysine supplementation in cats with enzootic upper respiratory disease (URD), 50 cats were fed a ration containing 11 or 51 g lysine/kg diet for 52 days. Food intake, body weight, clinical signs, plasma amino acid concentrations and presence of Chlamydophila felis or feline herpesvirus (FHV)-1 DNA within the conjunctival fornix were assessed. Food and lysine intake of both dietary groups decreased between days 17 and 22, coinciding with peak disease and viral presence. Mean disease score for cats fed the supplemented ration (0.94) was higher than for those fed the basal diet (0.21); however, this could be attributed to a small subset of male cats which demonstrated fighting behavior that may have contributed to stress within that cage. FHV-1 DNA was detected on 12 occasions in six cats receiving the supplemented diet and on one occasion in one cat fed the basal diet. C felis DNA was never detected. Mean plasma arginine concentration was lower and plasma lysine concentration was higher in supplemented cats. Mean plasma arginine concentration declined throughout the study in both dietary groups. Data from the present study raise important questions but do not permit a definitive conclusion regarding the efficacy of dietary lysine supplementation in cats with enzootic URD.