A critical role for the innate immune signaling molecule IRAK-4 in T cell activation

IRAK-4 is a protein kinase that is pivotal in mediating signals for innate immune responses. Here, we report that IRAK-4 signaling is also essential for eliciting adaptive immune responses. Thus, in the absence of IRAK-4, in vivo T cell responses were significantly impaired. Upon T cell receptor stimulation, IRAK-4 is recruited to T cell lipid rafts, where it induces downstream signals, including protein kinase C activation through the association with Zap70. This signaling pathway was found to be required for optimal activation of nuclear factor kappaB. Our findings suggest that T cells use this critical regulator of innate immunity for the development of acquired immunity, suggesting that IRAK-4 may be involved in direct signal cross talk between the two systems.     

Effect of bovine lactoferrin on the immune responses of captive bottlenosed dolphins (Tursiops truncatus) being transported over long distances

Bovine lactoferrin was administered orally, in feed, to six bottlenosed dolphins (Tursiops truncatus) before they were transported for approximately six hours; their stress responses were compared with those of five untreated dolphins. During the journey the dolphins had an increased plasma concentration of cortisol, and lymphopenia, eosinopenia and mild neutrophilia, indicating a stress response. The administration of lactoferrin did not affect the function of the dolphins' polymorphonuclear leucocytes, but affected their leucogram by maintaining the number of circulating eosinophils.     

Comparative assessment of Th1 and Th2 cytokines of swamp type buffalo and other bubaline breeds by molecular cloning, sequencing and phylogenetics

Comparative assessment of Th1 and Th2 cytokines of three bubaline breeds namely swamp buffalo, its crossbreed with riverine buffalo (CB), and the improved breed of Bulgarian Murrah buffalo (BMB), was done by molecular cloning, sequencing and phylogenetic analysis. The Th1 cytokines analyzed included IL-2, IL-12p35, IL-12p40, and IFN-gamma while Th2 cytokines included IL-4 and IL-10. Both groups showed strict conservation in the putative secondary structures and amino acid residues within the tribe Bovini, which indicated functional cross-reactivity. Nucleotide sequence homology ranged from 98.6 to 100.0% and was lowest for IL-12p35. With regard to amino acid sequence, the lowest homology was observed in IL-4 with 97.8%. This substitution was mainly due to differences in mRNA splicing. The phylogenetic relationship of the buffalo breeds was analyzed and showed them as a cluster comprised mainly of species belonging to the order Artiodactyla, including cattle and pigs. A deeper knowledge of these cytokine structures will favor understanding of water buffalo immunology and how much it differs from its closest subspecies and other animals.     

iRhom2 regulation of TACE controls TNF-mediated protection against Listeria and responses to LPS

Innate immune responses are vital for pathogen defense but can result in septic shock when excessive. A key mediator of septic shock is tumor necrosis factor-α (TNFα), which is shed from the plasma membrane after cleavage by the TNFα convertase (TACE). We report that the rhomboid family member iRhom2 interacted with TACE and regulated TNFα shedding. iRhom2 was critical for TACE maturation and trafficking to the cell surface in hematopoietic cells. Gene-targeted iRhom2-deficient mice showed reduced serum TNFα in response to lipopolysaccharide (LPS) and could survive a lethal LPS dose. Furthermore, iRhom2-deficient mice failed to control the replication of Listeria monocytogenes. Our study has identified iRhom2 as a regulator of innate immunity that may be an important target for modulating sepsis and pathogen defense.     

Increased bovine Tim-3 and its ligand expressions during bovine leukemia virus infection

ABSTRACT: The immunoinhibitory receptor T cell immunoglobulin domain and mucin domain-3 (Tim-3) and its ligand, galectin-9 (Gal-9), are involved in the immune evasion mechanisms for several pathogens causing chronic infections. However, there is no report concerning the role of Tim-3 in diseases of domestic animals. In this study, cDNA encoding for bovine Tim-3 and Gal-9 were cloned and sequenced, and their expression and role in immune reactivation were analyzed in bovine leukemia virus (BLV)-infected cattle. Predicted amino acid sequences of Tim-3 and Gal-9 shared high homologies with human and mouse homologues. Functional domains, including tyrosine kinase phosphorylation motif in the intracellular domain of Tim-3 were highly conserved among cattle and other species. Quantitative real-time PCR analysis showed that bovine Tim-3 mRNA is mainly expressed in T cells such as CD4+ and CD8+ cells, while Gal-9 mRNA is mainly expressed in monocyte and T cells. Tim-3 mRNA expression in CD4+ and CD8+ cells was upregulated during disease progression of BLV infection. Interestingly, expression levels for Tim-3 and Gal-9 correlated positively with viral load in infected cattle. Furthermore, Tim-3 expression level closely correlated with up-regulation of IL-10 in infected cattle. The expression of IFN-γ and IL-2 mRNA was upregulated when PBMC from BLV-infected cattle were cultured with Cos-7 cells expressing Tim-3 to inhibit the Tim-3/Gal-9 pathway. Moreover, combined blockade of the Tim-3/Gal-9 and PD-1/PD-L1 pathways significantly promoted IFN-γ mRNA expression compared with blockade of the PD-1/PD-L1 pathway alone. These results suggest that Tim-3 is involved in the suppression of T cell function during BLV infection.     

Prevalence of extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae in food-producing animals

To evaluate the diversity of extended-spectrum β-lactamases (ESBL) genes among food-producing animals, 48 isolates of ESBL-producing Escherichia coli isolates were obtained from rectal samples of broilers, layers, beef cattle and pigs, at the slaughterhouse level. ESBL-carrying E. coli were isolated from 60.0% of individual broiler rectal samples, 5.9% of layers, 12.5% of beef cattle and 3% of pigs. One ESBL-producing Klebsiella pneumoniae was isolated from a broiler. The ESBL-positive E. coli isolates from broilers harbored various ESBL genes: bla (SHV-12), bla(CTX-M-2), bla(CTX-M-14), bla(CTX-M-15) and bla(CTX-M-44). The plasmid DNAs were analyzed by restriction patterns. Homogeneous band patterns were yielded in those of K. pneumoniae and E. coli isolates harboring the bla(CTX-M-2) gene from different farms. No genetic relation between the 2 CTX-M-14 ESBL-producing strains was found by pulsed-field gel electrophoresis, although 2 plasmids in these strains, obtained from different broiler farms, were similar to each other. This study provides evidence that the proliferation of CTX-M-producing E. coli is due to the growth of indigenous CTX-M-producing strains and the possible emergence of strains that acquired CTX-M genes by horizontal transfer in different broiler farms. CTX-M-producing coliforms in broilers should be controlled due to the critical importance of cephalosporins and the zoonotic potential of ESBL-producing bacteria.     

PCR-based detection of blood parasites in cattle and adult Rhipicephalus appendiculatus ticks

To ascertain the infection rate for tick-borne pathogens in Zambia, an epidemiological survey of Theileria parva, Babesia bigemina and Anaplasma marginale in traditionally managed Sanga cattle was conducted using PCR. Of the 71 native Zambian cattle, 28 (39.4%) were positive for T. parva, 16 (22.5%) for B. bigemina and 34 (47.9%) for A. marginale. The mixed infection rate in cattle was 8.5% (6/71), 16.9% (12/71), 7.0% (5/71) and 2.8% (2/71) for T. parva/B. bigemina, T. parva/A. marginale, B. bigemina/A. marginale and T. parva/B. bigemina/A. marginale, respectively.To predict the risk for transmission of tick-borne pathogens from ticks to cattle, a total of 74 Rhipicephalus appendiculatus ticks were collected from a location where cattle had been found positive for T. parva. Of the ticks collected, 10 (13.5%) were found to be PCR-positive for T. parva. The results suggest that the infection rate for tick-borne pathogens was relatively high in Sanga cattle and that adult R. appendiculatus ticks were highly infected with T. parva.     

Measurement of plasma chromogranin A concentrations for assessment of stress responses in dogs with insulin-induced hypoglycemia

OBJECTIVE: To determine whether cross-reactivity exists between canine chromogranin A (CgA) and anti-human CgA antibody and investigate the usefulness of plasma CgA concentration measurements as an index of acute stress responses in dogs. ANIMALS: 12 healthy Beagles. PROCEDURE: Canine CgA was extracted and purified from canine adrenal glands of cadaver dogs for studying cross-reactivity with anti-human CgA antibody. Western blotting with anti-human CgA antibody was performed. Blood samples were collected from dogs at 0, 10, 20, 30, 40, 60, 120, and 180 minutes after IV administration of saline (0.9% NaCl) solution or insulin. Canine plasma CgA concentrations were determined by use of a CgA ELISA kit with rabbit antiserum against the carboxy-terminal fragment of human CgA. Plasma cortisol and catecholamine (ie, norepinephrine and epinephrine) concentrations were measured by use of an ELISA and a high-performance liquid chromatography method, respectively. RESULTS: Purified canine CgA was specifically detected by use of western blot analysis and an ELISA with anti-human CgA antibody. An increase in plasma CgA concentrations was observed in insulin-induced hypoglycemic dogs. Changes in plasma CgA concentration were correlated with changes in plasma cortisol or catecholamine concentrations of hypoglycemic dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Use of the CgA ELISA kit for determination of human plasma CgA concentrations is applicable to the measurement of canine plasma CgA concentrations. Canine plasma CgA concentrations, along with measurements of plasma cortisol and catecholamine concentrations, correctly reflect insulin-induced hypoglycemic stressed conditions in dogs. Measurement of canine plasma CgA concentrations may provide a useful index for evaluation of an acute stress response.     

Multiple perineuriomas in chicken (Gallus gallus domesticus)

Intraneural perineurioma is an extremely rare condition characterized by perineurial cell proliferation within peripheral nerve (PN) sheaths. In the veterinary field, this entity has been reported only in a dog. We examined multiple enlargements of PNs in 11 chickens (Gallus gallus domesticus) (9 Japanese bantams and 2 specific pathogen-free White Leghorn), which were inoculated with an avian leukosis virus (ALV) causing so-called fowl glioma. All chickens clinically exhibited progressive leg paralysis. Lumbosacral plexus, brachial plexus, and/or spinal ganglion were commonly affected, and these nerves contained a diffuse proliferation of spindle cells arranged concentrically in characteristic onion bulb-like structures surrounded by residual axons and myelin sheaths. The spindle cells were immunohistochemically negative for S-100alpha/beta protein. Electron microscopy revealed that these cells were characterized by short bipolar cytoplasmic processes, occasional cytoplasmic pinocytotic vesicles, and discontinuous basal laminae. These features are consistent with those of intraneural perineurioma. Furthermore, the specific sequence of the ALV was detected in the PN lesions of 8/11 (73%) birds by polymerase chain reaction. These results indicate that the multiple intraneural perineuriomas of chicken may be associated with the ALV-A causing fowl glioma.     

Effect of nutritional conditions on changes in leukocyte populations in Japanese black calves

To clarify the effect of nutritive conditions on changes in immune cells in Japanese Black (JB) calves during the growth period, leukocyte populations were analyzed in ten healthy JB calves managed in one herd. The calves were divided into two groups: five calves in Group 1 were given insufficient nutrition, and the other five calves in Group 2 received adequate nutrition. The levels of serum total cholesterol and glucose were significantly lower in Group 1 than in Group 2 at 1 month. The numbers of CD3+, CD4+ and CD8+ cells tended to be lower in Group 1 than in Group 2 at months 1 and 2, and the difference in CD4+ was significant at month 2. The number of MHC class-II(+high) cells was significantly lower in Group 1 than in Group 2 at months 1 and 2. These results suggest that adequate nutrition might stimulate an increase in immune cells in calves during the growth period.