Evidence for defective neutrophil function in lungs of calves exposed to infectious bovine rhinotracheitis virus

Calves were exposed to an aerosol of infectious bovine rhinotracheitis (IBR) virus followed five days later by an aerosol of Pasteurella haemolytica. The animals were subjected to bronchoalveolar lavage before IBR and four days after, and again at 0, 4, 24, and 48 hours following Pasteurella haemolytica challenge. The results of these experiments suggest that neutrophil infiltration into the lung, in response to the presence of the bacteria was delayed thereby allowing the bacteria to become established in the lung. Neutrophils in infected animals displayed little random migration in vitro and did not respond to a chemotactic stimulus. It was also found that alveolar macrophages from virus-infected animals were not able to produce neutrophil chemotactic factors. These data suggest that the decrease in neutrophil chemotaxis and the lack of chemotactic factor production by the alveolar macrophage following infection with infectious bovine rhinotracheitis virus may predispose infected cattle to a secondary bacterial infection.     

Isolation, characterization and in vitro mitogenic stimulation of peripheral blood lymphocytes from an American buffalo.

A rapid and reproducible method is described for the isolation and characterization of leukocytes from the peripheral blood of an American buffalo (bison). Centrifugation of the buffy coat cells on a Percoll gradient (1.079 g/mL) at 650 x g for 20 min resulted in the separation and high yields of pure viable leukocytes. The sheep erythrocyte-rosetting technique (ER) showed that 59% of the cells were ER+ (T lymphocytes). Fluorescein isothiocyanate (FITC)-conjugated peanut agglutinin and FITC-conjugated concanavalin A revealed 77% and 89% positive cells, respectively. The isolated leukocytes contained adherent accessory cells and functionally active T and B lymphocytes which proliferated in response to both T and B cell mitogens and to exogenous recombinant bovine interleukin-2 in the absence and/or presence of the thiol compound 2-mercaptoethanol.     

Bovine toll-like receptor 9: a comparative analysis of molecular structure, function and expression

Non-methylated CpG motifs, present in viral and bacterial DNA, are one of many pathogen-associated molecular patterns (PAMP) recognized by the mammalian innate immune system. Recognition of this PAMP occurs through a specific interaction with toll-like receptor 9 (TLR9) and this interaction can induce cytokine responses that influence both innate and adaptive immune responses. Previous investigations determined that both the flanking sequences in synthetic CpG oligodeoxynucleotides (CpG ODN) and the cellular pattern of TLR9 expression can influence species-specific responses to CpG ODN. Therefore, the structure, function and cellular distribution of bovine TLR9 were compared with what is known for mice and human. Analysis of the bovine TLR9 gene revealed greater sequence homology between cattle and humans than cattle and mice Similar CpG motifs induced optimal activation of both human and bovine leukocytes and these motifs were distinct from those which activated mouse leukocytes. Functional analyses with CpG ODN stimulated bovine blood leukocytes revealed that class A CpG ODN were more potent inducers of interferon-alpha (IFN-alpha) than class B CpG ODN. Furthermore, magnetic activated cell sorting of bovine blood leukocyte subpopulations implicated dendritic cells but not monocytes in the regulation of CpG ODN-induced IFN secretion. Thus, the cellular pattern of CpG ODN-induced responses in cattle shared many similarities with human leukocytes. Collectively, these analyses revealed substantial conservation of TLR9 structure and TLR9 function in blood leukocytes of humans, cattle and other domestic species.     

Formulation with CpG ODN enhances antibody responses to an equine influenza virus vaccine

Previous studies have shown that protection against equine influenza virus (EIV) is partially mediated by virus-specific IgGa and IgGb. In this study we tested whether addition of a CpG ODN formulation to a commercial killed virus vaccine would enhance EIV-specific IgGa and IgGb antibody responses, and improve protection against an experimental EIV challenge. Thirty na?ve horses were assigned to one of three groups and vaccinated as follows: 10 were given vaccine (Encevac TC4, Intervet Inc.) alone, 10 were given vaccine plus 0.25 mg CpG ODN 2007 formulated with 30% Emulsigen (CpG/Em), and 10 controls were given saline. All horses were challenged with live virus 12 weeks after the final vaccination. Antibody responses were tested by single radial hemolysis (SRH) and ELISA, and protection was evaluated by determination of temperature, coughing, and clinical scores. Killed virus vaccine combined with CpG/Em induced significantly greater serologic responses than did the vaccine alone. All antibody isotypes tested increased after the addition of CpG/Em, although no shift in relative antibody isotypes concentrations was detected. Vaccination significantly improved protection against challenge but the differences between the two vaccine groups were not statistically significant. This study is the first demonstration that CpG/Em enhances antigen-specific antibody responses in horses and supports its potential to be used as an adjuvant for vaccines against equine infections.     

Vaccination: a management tool in veterinary medicine

Conventional vaccines have been used for some 200 years, primarily to control infectious diseases. It is envisaged that such vaccines will continue to be used and new ones developed using conventional technology. However, in addition to conventional vaccines, novel approaches using biotechnology are already in use and many more are in various stages of development. These novel vaccines are not only being used to control infectious diseases, but also to improve productivity of livestock by modulating hormones, for gender selection, as well as in controlling ectoparasites. The recent developments in vaccination technology in all of these areas are described.     

Comparative virulence of different bovine rotavirus isolates.

Intestinal loops, ligated in colostrum-deprived calves were used to compare the virulence of four isolates of bovine rotavirus. Histopathological studies were carried out on infected and control loops and measurements of villous length, crypt depth, villus:crypt ratio and crypt mitotic index were recorded. Pathological changes associated with the rotaviruses included villous atrophy, flattening of absorptive epithelium and reduced villus:crypt ratios. The changes were confined to infected intestinal loops in which the presence of virus was demonstrated by specific immunofluorescence. Consistent differences in the measured histopathological changes suggested differences in virulence among the rotavirus isolates tested. The least virulent rotavirus isolate had a polypeptide electrophoretic pattern that differed from the other three more virulent isolates.     

Induction, regulation and physiological role of IL-17 secreting helper T-cells isolated from PBMC, thymus, and lung lymphocytes of young pigs

Interleukin-17 (IL-17) producing cells, referred to as Th17, have recently emerged as a third subset of the T helper (Th) cell family. Studies in mice have demonstrated that Th17 cells and their associated cytokines are involved in several autoimmune diseases and host defense against infection. Murine Th17 cells differentiate from naïve CD4⁺ T-cells in the presence of TGFβ and IL-6, however, there are contradicting reports as to the role of TGFβ in the differentiation of human Th17 cells and very little is known about these cells in other animals. We report here the presence of IL-17 secreting lymphocytes in the lung and peripheral blood of pigs. The cDNA of porcine IL-17 gene was cloned and sequenced from activated lung lymphocytes and PBMC from piglets. A 17 kDa recombinant protein was expressed and purified both under denaturing and native conditions from E. coli BL21 cells. Furthermore, we demonstrate that TGFβ in the presence of IL-6 and/or IL-1β induces in vitro differentiation of Th17 cells from naïve porcine CD4⁺ thymocytes.     

Evaluation of von Willebrand Factor During Pregnancy,Lactation and Oestrous Cycle in Bitches Affected and Unaffected by von Willebrand Disease

Plasmatic concentrations of von Willebrand Factor (vWF) increase during pregnancy in humans and dogs; however the mechanism of such increase is still not well defined. The aims of this study were: (i) to evaluate changes in vWF concentration during pregnancy and during the subsequent oestrous cycle in bitches affected and unaffected by von Willebrand Disease (vWD); (ii) to correlate the vWF levels and cortisol levels in both groups. Seven vWD affected (GI) and nine unaffected (GII) bitches were used. The animals were assessed during pregnancy, parturition, lactation and non‐gestational oestrous cycle in 11 moments (Pregnancy 1, Pregnancy 2, Parturition, Lactation 1, Lactation 2, Lactation 3, Anestrus, Proestrus, Oestrus, Diestrus 1, and Diestrus 2). The following tests were performed; measurement of von Willebrand factor antigen (vWF:Ag), albumin and cortisol. In both groups, vWF concentration remained stable during the non‐gestational oestrous cycle, but increased during pregnancy, with the highest value observed at parturition. Increases of 70% and 124% in vWF were seen in GI and GII, respectively, compared to anestrus. No correlation was found between vWF and cortisol. Values of vWF:Ag changed during pregnancy, with a peak at parturition, both in vWD affected and unaffected animals. Values of vWF were not altered in the different phases of the oestrous cycle following pregnancy in both groups. Evaluation of vWF during pregnancy can cause false negative results for vWD, but assessment can be performed at any point in the oestrous cycle of non‐pregnant bitches.     

Biological activity of immunostimulatory CpG DNA motifs in domestic animals

Bacterial DNA contains a much higher frequency of CpG dinucleotides than are present in mammalian DNA. Furthermore, bacterial CpG dinucleotides are often not methylated. It is thought that these two features in combination with specific flanking bases constitute a CpG motif that is recognized as a "danger" signal by the innate immune system of mammals and therefore an immune response is induced when these motifs are encountered. These immunostimulatory activities of bacterial CpG DNA can also be achieved with synthetic CpG oligodeoxynucleotides (ODN). Recognition of CpG motifs by the innate immune system requires engagement of Toll-like receptor 9 (TLR-9), which induces cell signaling and subsequently triggers a pro-inflammatory cytokine response and a predominantly Th1-type immune response. CpG ODN-induced innate and adaptive immune responses can result in protection in various mouse models of disease. Based on these observations, clinical trials are currently underway in humans to evaluate CpG ODN therapies for cancer, allergy and infectious disease. However, potential applications for immunostimulatory CpG ODN in species of veterinary importance are just being explored. In this review, we will highlight what is presently known about the immunostimulatory effects of CpG ODN in domestic animals.